RAGE Fusion Proteins with Improved Stability and Ligand Binding Affinity and Uses Thereof

ABSTRACT

The present invention provides soluble RAGE-Fc fusion proteins with increased stability and extended half-life capable of binding endogenous RAGE ligands with high apparent affinity. The present invention also provides methods of making and using stable, soluble RAGE-Fc fusion proteins. These soluble RAGE-Fc fusion proteins are useful as therapeutics based on their ability to bind endogenous RAGE ligands.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/731,663, filed Sep. 14, 2018, which is hereby incorporated byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Oct. 11, 2019, is named44263US_CRF_sequencelisting.txt and is 228,990 bytes in size.

BACKGROUND

The genes encoding both the bovine and human forms of receptor foradvanced glycation end-products (RAGE) were reported in 1992. The openreading frame (ORF) consisted of 404 amino acid residues organized into(from N to C terminus) a projected signal sequence of 22 amino acidresidues, an N-terminal exodomain of ˜321 residues, a transmembranedomain of 19 residues, and an intracellular domain of 41 residues. Theexodomain was shown to have three immunoglobulin (Ig)-like domains,including a variable domain and two constant regions. The signalsequence is thought to be residues 1-22, followed by the variable domainat residues 23-116, followed by a very short intervening sequence ofabout 6-8 residues leading to the C1 domain at residues 124-221. The C1and C2 domains are separated by a longer ˜18 residue linker. C2 spansresidues 239-304, followed by a highly flexible stem of ˜38 residuesthat allows for significant range of motion of the receptor on thesurface of the cell. The transmembrane domain is ca. 19 residues and theC-terminal intracellular portion of the protein spans residues 264-404,with a serine phosphorylation site at S391.

Multiple RAGE receptors may interact and form clusters, which may aid inthe binding of certain ligands, such as advanced glycation end products(AGEs), and result in intracellular signaling. Binding of a RAGE ligandto cell bound RAGE can trigger a series of downstream signaling events.Specific signaling profiles can differ, depending on the nature ofligand interaction, RAGE density, and other factors. Signaling mayinvolve phosphorylation of RAGE at amino acid residue S391 by proteinkinase C-zeta (PKCζ).

Nonenzymatic glycation and oxidation of proteins, lipids, and nucleicacids generates advanced glycation endproducts (AGEs), which arecanonical RAGE ligands.

In addition to AGE, RAGE binds multiple ligands including amyloid-beta,S100B, S100A1, S100A2, S100A7 (psoriasin), S100A11, S100A12, HMGB1(amphoterin), lipopolysaccharide (LPS), oxidized low-density lipoprotein(oxLDL), CD11b (MAC1), phosphatidyl serine, C3 a, S100P, S100G, S100Z,carbonylated proteins, malondialdehyde (MDA), laminin, type I Collagen,type IV Collagen, CAPZA1, CAPZA2, DDOST, LGALS3, MAPK1, MAPK3, PRKCSH,S100A4, S100A5, S100A6, S100A8, S100A9, S100P, and SAA1.

Accumulation of AGE leading to activation of RAGE has been implicated ina variety of diseases and disorders, including diabetes and itsmicrovascular complications, macrovascular complications, and othercomplications. AGEs and other RAGE ligands have been implicated in agingas well in a number of other diseases, including neurodegenerativedisease, diabetic complications, ischemia-reperfusion injury in multipleorgans, renal disease, etc. Soluble forms of RAGE (sRAGE and esRAGE)that include the extracellular ligand binding domain but lack thetransmembrane and cytoplasmic domains of the endogenous protein may beuseful for binding RAGE ligands, thereby impeding RAGE activation anddownstream signaling cascades. Thus, there exists a need for drug-likesoluble RAGE molecules with enhanced binding affinity to RAGE ligandsand an extended half-life suitable for therapeutic applications.Production of therapeutic proteins on a commercial scale requiresproteins that can be efficiently expressed and purified withoutdisrupting protein function. Manufacturability can be described as theability to express and purify a protein in a sufficiently efficientmanner and with sufficient stability and structural integrity to allowfor cost-effective production of the protein. For commercial purposes,manufacturability must be determined for each potential therapeuticprotein. Although protein expression and purification processes can beoptimized for a protein, manufacturability may be a function ofintrinsic properties of the protein.

SUMMARY OF THE INVENTION

The present disclosure provides biologically active therapeutic proteinsbased on RAGE having improved manufacturability properties capable ofefficient production as well as enhanced ligand binding properties andenhanced stability in vivo.

Disclosed here are compositions comprising RAGE fusion and methods ofuse thereof. Accordingly, one embodiment of the disclosure is anisolated polypeptide comprising a first domain and a second domain. Insome embodiments the first domain is at least 97% identical to thesequence of SEQ ID NO: 74. In some embodiments the second domaincomprises an Fc region of an immunoglobulin. In some embodiments thecarboxy terminus of the first domain is coupled to the amino terminus ofthe second domain by a peptide linkage.

In some embodiments the polypeptide is resistant to cleavage by adisintegrin and metalloproteinase 10 (ADAM 10). In some embodiments thepolypeptide is at least 15% more resistant to cleavage by at least oneof ADAM10, matrix metalloproteinase 9 (MMP9), and trypsin as compared toa polypeptide comprising the sequence set forth in SEQ ID NO: 5. In someembodiments the percent resistance equals the difference between thefraction of polypeptide that remains full length following incubationwith at least one of ADAM10, MMP9, and trypsin for a defined time periodcompared to a control polypeptide treated for the same time and underthe same conditions.

In some embodiments the polypeptide is resistant to degradation in humanserum. In some embodiments the polypeptide is at least 15% moreresistant to degradation in human serum as compared to a polypeptidecomprising the sequence set forth in SEQ ID NO: 5. In some embodimentsthe percent resistance equals the difference between the fraction ofpolypeptide that remains full length following incubation in human serumfor a defined time period as compared to a control polypeptide treatedfor the same time and under the same conditions.

In some embodiments the polypeptide has improved resistance to thermaldenaturation. In some embodiments the polypeptide has a higher onset ofthermal denaturation (T_(agg)) of at least 5° C. as compared to apolypeptide comprising the sequence set forth in SEQ ID NO: 5. In someembodiments the change in onset of thermal denaturation (T_(agg)) equalsthe temperature at which the polypeptide transitions from a compactfolded monomeric state to an unfolded state as analyzed in a definedtemperature gradient as compared to a control polypeptide treated in thesame temperature gradient and under the same conditions.

In some embodiments the polypeptide specifically binds at least one of:an advanced glycation endproduct (AGE), CML-HSA (carboxymethylated humanserum albumin), HMGB1 (amphoterin), amyloid-beta, S100A1, S100A2, S100A4(metastasin), S100A5, S100A6, S100A7 (psoriasin), S100A8/9, S100A11,S100A12, S100B, S100P, lipopolysaccharide (LPS), oxidized low-densitylipoprotein (oxLDL), CD11b (MAC1), phosphatidyl serine, C3a, S100P,S100G, S100Z, carbonylated proteins, malondialdehyde (MDA), laminin,type I Collagen, type IV Collagen, CAPZA1, CAPZA2, DDOST, LGALS3, MAPK1,MAPK3, PRKCSH, S100A4, S100A5, S100A6, S100A8, S100A9, S100P, and SAA1.

In some embodiments the polypeptide comprises a polypeptide dimer.

In some embodiments the first domain comprises at least one asparagineresidue linked to a glycan. In some embodiments the first domain anamino acid substitution at one or more of amino acid residues 3 or 59,wherein said amino acid residues 3 or 59 correspond to an amino acid atposition 3 or 59 of said first domain. In a preferred embodiment theamino acid at position 3 of the domain is substituted with glutamic acidor glutamine. In another preferred embodiment the amino acid at position59 of the first domain is substituted with alanine, glutamic acid, orglutamine. In one embodiment the amino acid residue at position 60 ofthe first domain is substituted with serine. In some embodiments thefirst domain comprises the sequence set forth in SEQ ID NO: 74.

In some embodiments the heavy chain of the polypeptide comprises CH2 andCH3 domains of a human IgG. In one embodiment the CH2 and CH3 domainscomprise the amino acid sequence set forth in SEQ ID NO: 4.

In some embodiments the immunoglobulin Fc of the polypeptide comprisesone or more amino acid substitutions at one or more of amino acidresidues 252, 254, or 256, numbered according to the EU numbering. Insome embodiments amino acid residue 252 is substituted with tyrosine. Insome embodiments amino acid residue 254 is substituted with threonine.In some embodiments amino acid residue 256 is substituted with glutamineor glutamic acid.

In some embodiments of the present disclosure the polypeptide maycomprise a Fc region of an IgG1, IgG2, or IgG4 immunoglobulin. In someembodiments the polypeptide may comprise a peptide linkage thatcomprises at least a portion of an immunoglobulin hinge region. In someembodiments the peptide linkage may comprise at least a portion of thehinge region of IgG1, IgG2, or IgG4. In some embodiments the peptidelinkage may comprise an amino acid sequence having at least 95% sequenceidentity to the sequence set forth in SEQ ID NO: 11, SEQ ID NO: 10, orSEQ ID NO: 8.

In some embodiments the carboxy terminal lysine of the IgG4 CH2-CH3immunoglobulin domain is deleted comprising the sequences set forth inSEQ ID NO: 54 and SEQ ID NO: 55.

In some embodiments the polypeptide has a higher apparent bindingaffinity to a receptor for advanced glycation endproducts (RAGE) ligandcompared to a polypeptide comprising the sequence of SEQ ID NO: 5. Insome embodiments the apparent equilibrium dissociation constant (Kd) ofthe interaction between the polypeptide and its ligand may be 20nanomolar (nM) or less.

Exemplary embodiments include a polypeptide that is expressed a greateramount in CHO-3E7 cells than a polypeptide comprising the sequence setforth in SEQ ID NO: 5 when CHO-3E7 cells are transfected under otherwiseidentical defined conditions with nucleic acid plasmid encoding eitherpolypeptide. In a preferred embodiment the greater amount is at least5%. In another preferred embodiment the nucleic acid plasmid comprisesthe nucleic acid vector pTT5.

One embodiment of the disclosure is an isolated polypeptide comprising aRAGE polypeptide coupled to an Fc region of an immunoglobulin. In someembodiments the carboxy terminus of the RAGE polypeptide is coupled tothe amino terminus of the immunoglobulin Fc region by a peptide linkage.In some embodiments the peptide linkages comprise novel stem and hingeregions. In some embodiments the RAGE polypeptide comprises the aminoacid sequence set forth in SEQ ID NO: 2.

In some embodiments the polypeptide has the amino acid sequence of SEQID NO: 53. In some embodiments the polypeptide has the amino acidsequence of SEQ ID NO: 12. In some embodiments the polypeptide has theamino acid sequence of SEQ ID NO: 15. In some embodiments thepolypeptide has the amino acid sequence of SEQ ID NO: 16.

Some embodiments of the disclosure comprise an isolated nucleic acidmolecule comprising a polynucleotide encoding a polypeptide comprising aRAGE polypeptide coupled to a heavy chain fragment of an Fc region of animmunoglobulin. In some embodiments the polynucleotide encodes apolypeptide comprising a first amino acid sequence and a second aminoacid sequence. In some embodiments the sequence of the first domain isat least 97% identical to the sequence set forth in SEQ ID NO: 74. Insome embodiments the second amino acid sequence comprises an Fc regionof an immunoglobulin. In some embodiments the carboxy terminus of thefirst amino acid sequence is coupled to the amino terminus of the secondamino acid sequence by a peptide linkage. In some embodiments thepolynucleotide is operably linked to a transcriptional or translationalregulatory sequence.

A further embodiment comprises a vector comprising an isolated nucleicacid molecule comprising a polynucleotide encoding a polypeptidecomprising a RAGE polypeptide coupled to a heavy chain fragment of an Fcregion of an immunoglobulin. Some embodiments of the present disclosurecomprise a host cell comprising a vector comprising an isolated nucleicacid molecule comprising a polynucleotide encoding a polypeptidecomprising a RAGE polypeptide coupled to a heavy chain fragment of an Fcregion of an immunoglobulin. In some embodiments the host cell is amammalian cell.

An embodiment of the present disclosure comprises a therapeuticcomposition for treating a RAGE-mediated disorder wherein thecomposition comprises a first amino acid sequence and a second aminoacid sequence. In some embodiments the first domain is at least 97%identical to the sequence set forth in SEQ ID NO: 74. In someembodiments the second amino acid sequence comprises a heavy chainfragment of an Fc region of an immunoglobulin. In some embodiments thecarboxy terminus of the first amino acid sequence is coupled to theamino terminus of the second amino acid sequence by a peptide linkage.In some embodiments the peptide linkage linking the first amino acidsequence and the second amino acid sequence comprises a stem derivedfrom a soluble splice variant and a silent antibody hinge region.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1 is a schematic of a dimerized esRAGE-Fc fusion protein. The RAGEpolypeptide comprises V, C1, C2, and the stem domains. The Fcpolypeptide comprises the CH2 and CH3 domains. The linker between thetwo polypeptides is identified as the hinge.

FIGS. 2A-2L show expression of RAGE-Fc fusion protein constructsassessed by Western blot: Construct #1 (FIG. 2A); Construct #9 (FIG.2B); Construct #10 (FIG. 2C); Construct #11 (FIG. 2D); Construct #12(FIG. 2E); Construct #13 (FIG. 2F); Construct #14 (FIG. 2G); Construct#15 (FIG. 211); Construct #16 (FIG. 21); Construct #17 (FIG. 2J);Construct #18 (FIG. 2K); and Construct #19 (FIG. 2L).

FIGS. 3A-3J show expression of RAGE-Fc fusion protein constructsassessed by Western blot: Construct #20 (FIG. 3A); Construct #21 (FIG.3B); Construct #22 (FIG. 3C); Construct #23 (FIG. 3D); Construct #24(FIG. 3E); Construct #25 (FIG. 3F); Construct #26 (FIG. 3G); Construct#27 (FIG. 3H); Construct #28 (FIG. 31); and Construct #29 (FIG. 3J).

FIGS. 4A-4I show expression of RAGE-Fc fusion protein constructsassessed by Western blot: Construct #30 (FIG. 4A); Construct #31 (FIG.4B); Construct #32 (FIG. 4C); Construct #33 (FIG. 4D); Construct #34(FIG. 4E); Construct #35 (FIG. 4F); Construct #36 (FIG. 4G); Construct#16ΔK (FIG. 4H); Construct #12ΔK (FIG. 4I).

FIGS. 5A-5F show scaled-up expression of RAGE-Fc fusion proteinconstructs assessed by Western blot: Construct #1 (FIG. 5A); Construct#9 (FIG. 5B); Construct #10 (FIG. 5C); Construct #11 (FIG. 5D);Construct #12 (FIG. 5E); Construct #6 (FIG. 5F).

FIGS. 6A-6D show the concentration response curves generated by ELISAassays performed to assess ligand binding activities of RAGE-Fc fusionproteins: CML-HSA (FIG. 6A); HMGB1 (FIG. 6B); S100A9 (FIG. 6C); S100A12(FIG. 6D).

FIGS. 7A-7G show SDS-PAGE results of RAGE-Fc fusion proteins incubatedwith buffer alone for 0 and 24 hours (FIG. 7A); MMP9 for 0 and 24 hours(FIG. 7B); MMP9 for 15 and 24 hours (FIG. 7C); ADAM10 for 0 and 2 hours(FIG. 7D); ADAM10 for 15 and 24 hours (FIG. 7E); trypsin for 0 and 2hours (FIG. 7F); and trypsin for 15 and 24 hours (FIG. 7G).

FIGS. 8A-8D show time course proteolysis data for fusion proteinsincubated in the absence of protease (FIG. 8A); or in the presence ofMMP9 (FIG. 8B); ADAM10 (FIG. 8C); or trypsin (FIG. 8D).

FIGS. 9A-9D show SDS-PAGE results of RAGE-Fc fusion proteins incubatedwith human serum for 0 hours (FIG. 9A); 17 hours (FIG. 9B); 49 hours(FIG. 9C); and 138 hours (FIG. 9D).

FIG. 10 shows time course proteolysis data for fusion proteins incubatedin human serum over 138 hours.

FIGS. 11A-11D show thermal denaturation curves of RAGE-Fc fusionproteins as measured by dynamic light scattering: Construct #1 (FIG.11A); Construct #10 (FIG. 11B); Construct #12 (FIG. 11C); and Construct#16 (FIG. 11D).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure describes fusion proteins comprisingextracellular RAGE joined via a peptide linkage at the carboxyl terminuswith an immunoglobulin Fc. The fusion proteins of the disclosure arecharacterized by their ability to bind to at least one RAGE ligand(e.g., advanced glycation end-product (AGE), HMGB1 (amphoterin),S100A11, S100A12) with high affinity, thereby disrupting endogenousRAGE-mediated signaling. The RAGE fusion proteins of the presentdisclosure are further characterized by enhanced stability, extendedhalf-life, and improved manufacturability compared to other soluble RAGEproteins.

The stabilized RAGE-Fc fusion proteins are characterized by a RAGEprotein that is different from the extracellular domain of thefull-length RAGE polypeptide by the addition of 16 amino acids at thecarboxyl terminus. The carboxyl terminus of the RAGE protein is joinedto the amino terminus of a human immunoglobulin Fc via a peptide linkagecomprised of at least part of an immunoglobulin hinge. In someembodiments a short peptide linker may be inserted between the RAGEprotein and the immunoglobulin hinge.

DEFINITIONS

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

The term “ameliorating” refers to any therapeutically beneficial resultin the treatment of a disease state (e.g., a RAGE-mediated disease).

The term “isolated” refers to a protein or polypeptide molecule purifiedto some degree from endogenous material.

The term “RAGE” as used herein refers to the polypeptide sequenceencoding Receptor for Advanced Glycation Endproduct (RAGE) or anyvariation thereof, including, but not limited to, isoforms that lack allor part of the N-terminal V-type immunoglobulin domain (N-truncated),isoforms that lack all or part of the transmembrane domain(C-truncated), and isoforms that comprise 1, 2, 3, 4 or more than 4amino acid substitutions compared to wild-type RAGE.

The term “sRAGE” as used herein refers to soluble RAGE or RAGE lacking atransmembrane domain (C-truncated). As used herein, sRAGE refers tosoluble RAGE that is generated as a result of protease cleavage thatremoves the transmembrane domain.

The term “esRAGE” (endogenous soluble RAGE) as used herein refers tosoluble RAGE generated by an alternative splice site that results in amodified C-terminus comprising the following sequence at positions 332to 347: EGFDKVREAEDSPQHM (the C-terminal portion of the V1 stem) (SEQ IDNO: 52). As used herein, “esRAGE” may comprise one or more amino acidsubstitutions, including point mutations within amino acid positions 332to 347. The term percent “identity,” in the context of two or morenucleic acid or polypeptide sequences, refer to two or more sequences orsubsequences that have a specified percentage of nucleotides or aminoacid residues that are identical, when compared and aligned for maximumcorrespondence using BLASTP and BLASTN algorithms, using the defaultparameters as publicly available through the National Center forBiotechnology Information (www.ncbi.nlm.nih.gov/). Depending on theapplication, the percent “identity” can exist over a region of thesequence being compared, e.g., over a functional domain, or,alternatively, exist over the full length of the two sequences to becompared. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byvisual inspection (see generally Ausubel et al.).

As used herein, the terms “treatment,” “treating,” and the like, referto administering an agent, or carrying out a procedure for the purposesof obtaining an effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of effecting a partial or complete cure fora disease and/or symptoms of the disease. “Treatment,” as used herein,covers any treatment of any pathological state in a mammal, particularlyin a human, and includes: (a) inhibiting the disease, i.e., arrestingits development; (b) relieving the disease, i.e., causing regression ofthe disease; (c) delaying onset of the disease; (d) decreasing theduration of the disease; (e) relieving or reducing the severity of anysymptom of the disease; or (f) decreasing the risk or severity of anycomplication of the disease.

Treating may refer to any indicia of success in the treatment oramelioration or prevention of a pathologic state, including anyobjective or subjective parameter such as abatement; remission;diminishing of symptoms or making the disease condition more tolerableto the patient; slowing in the rate of degeneration or decline; ormaking the final point of degeneration less debilitating. The treatmentor amelioration of symptoms can be based on objective or subjectiveparameters, including the results of an examination by a physician.Accordingly, the term “treating” includes the administration of thecompounds or agents of the present invention to delay, to alleviate, orto arrest or inhibit development of the symptoms or conditionsassociated with the pathologic state. The term “therapeutic effect”refers to the reduction, elimination, prevention, delayed onset, oraccelerated resolution of the disease, symptoms of the disease, or sideeffects of the disease in the subject.

The term “prevent” as used herein refers to avoiding or averting theonset of a symptom or symptoms characteristic of one or more diseasestates.

The term “prophylaxis” as used herein refers to therapy given to preventor ameliorate symptoms of one or more disease states.

“In combination with”, “combination therapy” and “combination products”refer, in certain embodiments, to the concurrent administration to apatient of a first therapeutic and the compounds as used herein. Whenadministered in combination, each component can be administered at thesame time or sequentially in any order at different points in time.Thus, each component can be administered separately but sufficientlyclosely in time so as to provide the desired therapeutic effect.

The term “subject” refers to any animal, such as mammals, includinghumans.

The term “sufficient amount” means an amount sufficient to produce adesired effect, e.g., an amount sufficient to modulate proteinaggregation in a cell.

The term “therapeutically effective amount” is an amount that iseffective to ameliorate a symptom of a disease. A therapeuticallyeffective amount can be a “prophylactically effective amount” asprophylaxis can be considered therapy.

The term “percent resistant” refers to the percent resistance equal tothe difference between the fraction of peptide that remains full lengthfollowing incubation with at least one of ADAM10, MMP9, and trypsin fora defined time period compared to a control peptide treated for the sametime and under the same conditions.

The term “increased thermal stability” refers to the highest temperaturewhich a polypeptide remains in a folded state following incubation intemperature gradient for a defined time period as compared to a controlpolypeptide treated with the same temperature gradient and under thesame conditions.

The term “specific binding,” as used herein, refers to an affinitybetween a receptor and its ligand in which the K_(d) value is below10⁻⁶M, 10⁻⁷M, 10⁻⁸M, 10⁻⁹M, or 10⁻¹⁰M.

Abbreviations used in this application include the following: AdvancedGlycation Endproduct (AGE), Receptor for Advanced Glycation Endproduct(RAGE), soluble RAGE (sRAGE), endogenous secretory RAGE (esRAGE),immunoglobulin (Ig).

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

RAGE Fusion Proteins

The present disclosure provides RAGE fusion proteins, and methods ofmaking and using such fusion proteins.

In a first aspect, isolated polypeptides are provided.

Embodiments of the isolated polypeptides are fusion proteins comprisingfour modules: an amino-terminus derived from a RAGE exodomain, a stemderived from a soluble splice variant (esRAGE) or a shortened portion ofits stem region (lacking the C-terminal 13 amino acid residues of thestem containing the proteolytic cleavage site), a silent antibody hingeregion, and an antibody Fc region. In some embodiments the fusionprotein comprises an esRAGE polypeptide. The esRAGE polypeptide may beat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto SEQ ID NO: 74.

In typical embodiments, the isolated polypeptides comprise a firstdomain wherein said first domain has an amino acid sequence at least 97%identical to the sequence of SEQ ID NO:74; and a second domaincomprising a fragment of a Fc region of an immunoglobulin, wherein thecarboxy terminus of said first domain is coupled to the amino terminusof said second domain by a peptide linkage.

SEQ ID NO: 1 provides the sequence of esRAGE (including the N-terminalleader sequence) and SEQ ID NO:74 provides the sequence of mature esRAGE(lacking the N-terminal leader sequence). esRAGE is an endogenoussoluble form of RAGE generated by an alternative splice site whichresults in the extracellular domain of full RAGE, modified at thecarboxyl terminus by an additional 16 amino acids beginning at position332 (SEQ ID NO: 1) or position 310 (SEQ ID NO: 74).

In various embodiments, the first domain has a sequence that differsfrom SEQ ID NO: 1 by 1, 2, 3, 4, 5, 6, 7, or more than 7 amino acids. Insome embodiments, the first domain has a substitution of the asparagineat position 25 of SEQ ID NO: 1 (position 3 of SEQ ID NO: 74), whereinthe substitution is a glutamic acid or glutamine. In some embodiments,the first domain has the asparagine at position 81 of SEQ ID NO: 1(position 59 of SEQ ID NO: 74) substituted with alanine. In someembodiments, the first domain has the glycine at position 82 of SEQ IDNO: 1 (position 60 of SEQ ID NO: 74) substituted with serine. In someembodiments, the first domain has an amino acid inserted, deleted, orsubstituted in the amino acid sequence corresponding to positions332-347 of SEQ ID NO: 1 (positions 310-325 of SEQ ID NO: 74).

In some embodiments the fusion protein comprises a full-length RAGEpolypeptide.

In some embodiments the fusion protein comprises a RAGE polypeptide witha shortened stem region lacking the C-terminal 13 amino acid residues.The shortened stem RAGE polypeptide may be at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 74.

In some embodiments the amino-terminus module may comprise a signalsequence. The signal sequence may comprise the amino acid residues atpositions 1-22 of the amino acid sequence set forth in SEQ ID NO: 1. Insome embodiments the signal sequence may be at least 50%, 60%, 70%, 80%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to theamino acid sequence at positions 1-22 of the sequence set forth in SEQID NO: 1. In still other embodiments the amino-terminus module maycomprise any signal sequence useful for expressing RAGE fusion proteins.

In some embodiments, the amino-terminus module comprising a RAGEpolypeptide of the present disclosure may be glycosylated on at leastone of the asparagine residues at positions 25 and 81 (SEQ ID NO: 1) orpositions 3 and 59 (SEQ ID NO: 74). In some embodiments glycosylation ateither position may be required for optimal ligand binding. In someembodiments glycosylation of the asparagine residues at both position 25and 81 (SEQ ID NO: 1) or position 3 and 59 (SEQ ID NO: 74) may impairligand binding.

In some embodiments of the present disclosure the RAGE polypeptide maydimerize. In some embodiments the RAGE polypeptide may dimerize uponbinding a RAGE ligand. In some cases the V domains of RAGE polypeptidesmay interact to form homodimers. In some cases dimerization may bemediated by the C1 or C2 domains.

In some embodiments, the RAGE polypeptide may be linked to a polypeptidecomprising an immunoglobulin domain or a portion (e.g., a fragmentthereof) of an immunoglobulin domain. In some cases the polypeptidecomprising an immunoglobulin domain or a portion of an immunoglobulindomain may comprise a human IgG Fc region or a portion thereof. In somecases the human IgG Fc region comprises at least a portion of the CH2and CH3 domains of a human IgG Fc region. The human IgG Fc region may bederived from any of the known IgG subtypes: IgG1, IgG2, IgG3, or IgG4.

In some cases the RAGE fusion protein may comprise the CH2 and CH3domains of human IgG4. In some embodiments the fusion protein maycomprise the sequence set forth in SEQ ID NO: 7. In other embodimentsthe fusion protein may comprise a polypeptide having at least 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 7.

In some embodiments the Fc polypeptide of the fusion protein may beproinflammatory in vivo. In other embodiments, the Fc polypeptide may besilenced (e.g. comprise a peptide sequence that prevents formation ofimmune complexes that otherwise would form through productive engagement(i.e. engagement that results in an inflammatory response) of the Fcpolypeptide to an Fc receptor) in vivo. In some embodiments the Fcpolypeptide may be silenced with respect to binding Fc-gamma receptorsby the nature of specific AA sequences in the hinge region.

The Fc polypeptide of the RAGE fusion protein may increase the stabilityof the fusion protein. For example, the Fc polypeptide of the fusionprotein may contribute to stabilizing the RAGE fusion protein, therebyincreasing the half-life of the RAGE fusion protein. In some cases theFc polypeptide may significantly increase the serum half-life.

In some embodiments the RAGE fusion protein of the present disclosuremay be more stable than RAGE fusion proteins in the prior art becausethe RAGE fusion protein of the disclosure lacks protease cleavage sitesof RAGE fusion proteins of the prior art. For example, removal of theadditional 16 amino acids in the esRAGE splice variant may result in theelimination of one or more protease cleavage sites. In some embodimentsthe RAGE fusion protein lacks the C-terminal 13 amino acids of the RAGEstem and thereby lacks a protease cleavage site of the prior art. Insome embodiments the Fc polypeptide of the present disclosure mayinclude fewer protease cleavage sites than the prior art. In otherembodiments, the peptide linkage may include fewer protease cleavagesites than that in the prior art.

Protease cleavage sites are amino acid sequences recognized and cleavedby protease enzymes, resulting in a truncated polypeptide. Proteaseenzymes may include but are not limited to a disintegrin andmetalloproteinase 10 (ADAM10), matrix metalloproteinase 9 (MMP9), andtrypsin.

In one embodiment, the RAGE fusion protein of the present disclosurecomprises an Fc polypeptide optimized to increase the in vivo serumhalf-life of the fusion protein. In one embodiment the Fc polypeptide isoptimized by generating mutations (e.g., amino acid substitutions) thatincrease the half-life of the fusion protein. In one embodiment the Fcpolypeptide comprises mutations comprising amino acid substitutions atresidue positions 252, 254, and 256 (numbered according to the EU indexas in Kabat). In a preferred embodiment the residue at position 252 issubstituted with tyrosine, the residue at position 254 is substitutedwith threonine, and the residue at position 256 is substituted withglutamic acid (glutamate).

In some embodiments the serum half-life of the fusion protein isincreased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or200% as compared to a polypeptide comprising the sequence set forth inSEQ ID NO: 5.

The RAGE fusion protein of the present disclosure further comprises apeptide linkage (linker). Linkers serve primarily as a spacer between apolypeptide and a second heterologous polypeptide or other type offusion. In one embodiment the linker is made up of amino acids linkedtogether by peptide bonds, preferably from 1 to 20 amino acids linked bypeptide bonds, wherein the amino acids are selected from the 20naturally occurring amino acids. In one embodiment a linker is made upof a majority of amino acids that are sterically unhindered (e.g.,glycine, alanine). In a further embodiment the linker may comprise theamino acid sequence of an IgG hinge region or partial IgG hinge region,as exemplified in SEQ ID NO: 8.

Expression of RAGE Fusion Proteins

RAGE fusion proteins of the present disclosure may be produced using avariety of expression-host systems. These systems include but are notlimited to microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; and insect cell systemsinfected with virus expression vectors (e.g., baculovirus); andmammalian systems. Mammalian cells useful in recombinant proteinproduction include but are not limited to VERO cells, HeLa cells,Chinese hamster ovary (CHO) cells (e.g., CHO-3E7 cells), COS cells,W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562, L cells, C127 cells,HEK 293, epidermal A431 cells, human Colo205 cells, HL-60, U937, HaK,and Jurkat cells. Mammalian expression allows for the production ofsecreted or soluble polypeptides which may be recovered from the growthmedium.

Recombinant expression of a RAGE fusion protein of the presentdisclosure may require construction of a plasmid comprising apolynucleotide that encodes the fusion protein. The plasmid may begenerated by sub-cloning the polynucleotide into an expression vector(e.g. pTT5, pcDNA3.1) using standard recombinant techniques, wherein theexpression vector comprises regulatory signals for transcription andtranslation in mammalian systems.

In one embodiment a recombinant plasmid comprising a polynucleotide thatencodes the fusion protein may be introduced into CHO cells bytransfection such that the cells express the fusion protein. In oneembodiment, cells expressing the fusion protein may be selected andcloned to generate cell lines that stably express the fusion protein.For example, cells expressing the recombinant construct may be selectedfor plasmid-encoded neomycin resistance by applying the antibiotic G418to transfected cells. Individual clones may be selected and clonesexpressing high levels of the fusion protein as detected by Western Blotanalysis of the cell supernatant may be expanded.

The RAGE fusion proteins of the present disclosure may be purifiedaccording to protein purification techniques known to those of skill inthe art. For example, supernatant from a system which secretesrecombinant protein into culture may be concentrated using acommercially available protein concentration filter. In one embodimentthe supernatant may be applied directly to a suitable affinitypurification matrix. For example, a suitable affinity purificationmatrix may comprise a molecule (e.g. Protein A, AGE) bound to a support.In one embodiment the supernatant may be applied to an anion exchangeresin, for example, a matrix having pendant diethylaminoethyl (DEAE)groups. In another embodiment the supernatant may be applied to a cationexchange matrix. The matrices may include but are not limited to,acrylamide, agarose, dextran, and cellulose. After washing and elutingfrom the purification matrix, eluted fractions may be concentrated. Insome embodiments the elution may be subjected to aqueous ion exchange orsize exclusion chromatography. In some embodiments the elution may besubjected to high performance liquid chromatography (HPLC) for finalpurification.

Pharmaceutical Compositions

Methods for treatment of RAGE-mediated diseases are also encompassed bythe present disclosure. Said methods of the disclosure includeadministering a therapeutically effective amount of esRAGE-Fc fusionprotein. The fusion protein of the disclosure can be formulated inpharmaceutical compositions. These compositions can comprise, inaddition to one or more of the esRAGE-Fc fusion proteins, apharmaceutically acceptable excipient, carrier, buffer, stabilizer, orother materials well known to those skilled in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nature of the carrier or other materialcan depend on the route of administration, e.g. intravenous, cutaneousor subcutaneous, nasal, intramuscular, intraperitoneal routes.

For pharmaceutical compositions for intravenous, cutaneous orsubcutaneous injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilizers, buffers, antioxidants and/orother additives can be included, as required.

Administration of the pharmaceutically useful fusion protein of thepresent invention is preferably in a “therapeutically effective amount”or “prophylactically effective amount” (as the case can be, althoughprophylaxis can be considered therapy), this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofdisease being treated. Prescription of treatment, e.g. decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors, and typically takes account of the disorder to betreated, the condition of the individual patient, the site of delivery,the method of administration and other factors known to practitioners.Examples of the techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

A composition can be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Uses of RAGE Fusion Proteins

The present disclosure provides methods and pharmaceutical compositionsfor binding RAGE ligands with high affinity, thereby inhibiting orreducing RAGE activation and thus RAGE-mediated signaling. In oneaspect, the present disclosure provides methods and reagents fortreating RAGE-mediated disorders (e.g., inflammation, nephropathy,arteriosclerosis, retinopathy, and other complications resulting fromdiabetes) in a subject in need thereof by administering atherapeutically effective amount of the fusion proteins of thedisclosure to the subject. In one embodiment the fusion proteins of thepresent disclosure may bind one or more RAGE ligands in a subject andthereby decrease or inhibit RAGE-mediated signaling cascades. In someembodiments the fusion proteins may thereby reduce or inhibit aninflammatory response.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature.

Example 1: Expression and Purification of RAGE-IgG Fc Fusion Proteins.

The following methods were used for expressing and purifying the RAGE-Fcfusion proteins.

The following method was used to produce the RAGE-Fc protein comprisingthe amino acid sequence set forth in SEQ ID NO: 16. Polynucleotidesencoding esRAGE (SEQ ID NO: 1) were fused to polynucleotides encodingthe human IgG4 Fc (amino acid residues 359-590 of the amino acidsequence set forth in SEQ ID NO: 17) via polynucleotides encoding alinker sequence derived from the IgG2 hinge (SEQ ID NO: 9) by PCRoverlap extension. Primers used for PCR contained the mutation resultingin the amino acid substitutions of methionine to tyrosine at position252, serine to threonine at position 254, and threonine to glutamic acid(glutamate) at position 256 of the Fc polypeptide wherein the numberingis according to the EU index as in Kabat. The full polynucleotidesequence is SEQ ID NO: 43 for the RAGE-Fc fusion protein having theamino acid sequence set forth in SEQ ID NO:16. Double stranded DNAfragments were subcloned into pTT5 vector.

Transient expression of RAGE-Fc fusion proteins was carried out asfollows.

The RAGE-Fc polypeptide comprising the amino acid sequence set forth inSEQ ID NO: 16 was transiently expressed in CHO-3E7 cells grown inserum-free FreeStyle™ CHO Expression Medium (Thermo Fisher Scientific).The cells were maintained in Erlenmeyer Flasks (Corning Inc.) at 37° C.with 5% CO₂ on an orbital shaker (VWR Scientific). One day beforetransfection the cells were seeded at an appropriate density in CorningErlenmeyer Flasks. On the day of transfection, DNA containing apolynucleotide encoding the esRAGE-Fc polypeptide and transfectionreagent were mixed at an optimal ratio and then added into the flaskcontaining cells previously seeded for transfection. The recombinantplasmid DNA encoding the esRAGE-Fc polypeptide was transientlytransfected into suspension CHO-3E7 cell cultures. The cell culturesupernatant collected on post-transfection day 6 was used forpurification.

Purification of esRAGE-Fc fusion proteins was carried out as follows.

The cell culture broth was centrifuged and the resulting supernatant wasloaded onto a Monofinity A Resin prepacked affinity purification columnat an appropriate flow rate. After washing and elution with appropriatebuffer, the eluted fractions were pooled and buffer exchanged to finalformulation buffer.

The purified protein was analyzed by SDS-PAGE and Western blotting formolecular weight and purity measurements. Results of Western blots ofthe fusion proteins are shown in FIGS. 2A-4I: Construct #1 (FIG. 2A);Construct #9 (FIG. 2B); Construct #10 (FIG. 2C); Construct #11 (FIG.2D); Construct #12 (FIG. 2E); Construct #13 (FIG. 2F); Construct #14(FIG. 2G); Construct #15 (FIG. 2H); Construct #16 (FIG. 21); Construct#17 (FIG. 2J); Construct #18 (FIG. 2K); Construct #19 (FIG. 2L);Construct #20 (FIG. 3A); Construct #21 (FIG. 3B); Construct #22 (FIG.3C); Construct #23 (FIG. 3D); Construct #24 (FIG. 3E); Construct #25(FIG. 3F); Construct #26 (FIG. 3G); Construct #27 (FIG. 3H); Construct#28 (FIG. 31); Construct #29 (FIG. 3J); Construct #30 (FIG. 4A);Construct #31 (FIG. 4B); Construct #32 (FIG. 4C); Construct #33 (FIG.4D); Construct #34 (FIG. 4E); Construct #35 (FIG. 4F); Construct #36(FIG. 4G); Construct #16ΔK (FIG. 4H); Construct #12ΔK (FIG. 24I).

Expression of a number of fusion proteins was performed at 1L scale andproteins were purified by Protein A affinity chromatography, followed bySuperdex200 size exclusion chromatography. Purified protein was analyzedby SDS-PAGE and Western blotting for molecular weight and puritymeasurements. Results of Western blots of the fusion proteins are shownin FIG. 5: Construct #1 (FIG. 5A); Construct #9 (FIG. 5B); Construct #10(FIG. 5C); Construct #11 (FIG. 5D); Construct #12 (FIG. 5E); Construct#6 (FIG. 5F)

The lanes of each blot in FIGS. 2A-2L, FIGS. 3A-3J, FIGS. 4A-4I, andFIGS. 5A-5F are labeled according to the contents of each as follows:M_(2,) protein marker (GenScript, Cat. No. M00521); P, Human IgG1, Kappa(as positive control) (Sigma, Cat. No. 15154); 1, RAGE-Fc fusion proteinunder reducing conditions (with DTT); 2, RAGE-Fc fusion protein undernon-reducing conditions (no DTT). The primary antibody used for allblots was Goat Anti-Human IgG-HRP (GenScript, Cat. No. A00166).

The concentration of the purified protein was determined by Bradfordassay using bovine serum albumin (BSA) as a standard. Quantifiedexpression data is shown in Tables 9, 10 and 11.

Example 2: Assessing Binding Affinities of RAGE Fusion Proteins byELISA.

Functional ELISA assays were performed to assess the ligand bindingcharacteristics of RAGE-Fc fusion proteins. Apparent binding affinitiesof RAGE-Fc fusion proteins to the RAGE ligands CML-HSA, HMGB1, S100A9and S100A12 were measured for the following fusion proteins: Construct#1 (SEQ ID NO: 5), Construct #9 (SEQ ID NO: 53), Construct #10 (SEQ IDNO: 12), Construct #12 (SEQ ID NO: 15), and Construct #16 (SEQ ID NO:16). Previous experiments were carried out to determine fundamentalfunctionality of an ELISA, optimal coating concentrations and volumes, adynamic range for the RAGE-Fc constructs, as well as optimized antibodydilutions and TMB development times.

RAGE ligands CML-HSA, HMGB1, S100A9 and S100A12 were separately coatedonto a coated plate (MaxiSorp™) at a concentration of 50 nanomolar (nM),100 microliters (μL) per well. RAGE ligand CML-HSA was separately coatedonto a coated plate (MaxiSorp™) at a concentration of 100 nanograms(ng), 100 μL per well. The plates were then incubated overnight at 4° C.to allow the protein to bind to the plate coating. Following the coatingstep, the plates were washed once with 150 μL of wash solution (2.67 mMpotassium chloride, 1.47 potassium phosphate monobasic, 136.9 mM sodiumchloride, 8.10 mM sodium phosphate dibasic, 0.05% Tween-20). The platewas then aspirated and blocked for 90 minutes at 4° C. with 130 μL of asolution of 1% BSA (1 g/L) in DPBS (pH 7.4) with 0.03% sodium azide toprevent background binding to unfilled regions of the plate wells whileblocking with a protein that does not interact with soluble RAGEconstructs. After the blocking step, two washes were performed with thewash solution. The RAGE-Fc fusion protein was then incubated on thewells in log10 dilution with each separate ligand for 120 minutes at 37°C. while shaking. After the RAGE-Fc binding step, three washes wereperformed with the wash solution. Binding of the RAGE-Fc fusion toCML-HSA, HMGB1, S100A9, and S100A12 was detected with a horseradishperoxidase (HRP) conjugated antibody with antigen specificity to IgG Fc(Abcam, Cat. No. ab99759). 100 μL of antibody diluted 1:5000 in DBPS wasadded to the assay wells, followed by 60 minutes of incubation at 37° C.while shaking. The wells were then washed four times with the washsolution. 100 μL of TMB (ThermoFisher Scientific, Cat. No. 34029) wasthen added to each well. After approximately one minute, the reactionwas stopped by the addition of 50 μL of 1 M hydrochloric acid.Absorbance of the well contents was measured on a spectrophotometer at awavelength of 450 nM.

Results of the ELISA assays (FIGS. 6A-6D) show that the RAGE-Fc fusionproteins of the disclosure (Constructs #9, 10, 12, 16) bind to the RAGEligands CML-HSA (FIG. 6A), HMGB1 (FIG. 6B), S100A9 (FIG. 6C) and S100A12(FIG. 6D) with greater apparent affinity than the RAGE-Fc fusion in theprior art (Construct #1). Apparent Kd values were calculated for eachfusion protein-ligand interaction and are shown in Tables 1, 2, 3, and4.

TABLE 1 Apparent binding affinity of RAGE-Fc constructs to CML-HSAConstruct Construct Construct Construct Construct #1 #9 #10 #12 #16Apparent Kd 88 nM 6 nM 99 nM 39 nM 35 nM

TABLE 2 Apparent binding affinity of RAGE-Fc constructs to HMGB1Construct Construct Construct Construct Construct #1 #9 #10 #12 #16Apparent Kd 26 nM 2 nM 15 nM 7 nM 7 nM

TABLE 3 Apparent binding affinity of RAGE-Fc constructs to S100A9Construct Construct Construct Construct Construct #1 #9 #10 #12 #16Apparent Kd 266 nM 13 nM 41 nM 25 nM 27 nM

TABLE 4 Apparent binding affinity of RAGE-Fc constructs to S100A12Construct Construct Construct Construct Construct #1 #9 #10 #12 #16Apparent Kd 180 nM 9 nM 46 nM 44 nM 36 nM

Example 3: Assessing Susceptability to Proteolytic Degradation.

ADAM10 (a disintegrin and metalloproteinase 10) and MMP9 (matrixmetalloproteinase 9) are enzymes that cleave full length RAGE. Theenzymes were used to assess the vulnerability of RAGE-Fc fusion proteinsto proteolytic cleavage by biologically relevant enzymes. In addition,trypsin was used as a non-specific enzyme to assess the general proteaseresistance of each fusion protein. For comparison, the esRAGE-Fc fusionproteins of the present disclosure were tested against a purifiedversion identical to commercially available RAGE-Fc construct.

Each enzyme was verified to be functional under set assay conditions bydemonstrating cleavage of a known peptide substrate. In brief, 0.06 μMof ADAM10 or 0.01 μM of MMP9 was incubated with 5 μM of fluorogenicpeptide substrate [Mca-KPLGL-Dpa-AR-NH2 (SEQ ID NO: 75)]. Thefluorescence was measured kinetically at 320 nm excitation and 405 nmemission via an automated fluorescence microplate reader. Trypsin at0.002 μM was incubated with 766 μM of chromogenic substrate[Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride]. The absorbancewas measured kinetically at 405 nm via an automated microplatespectrophotometer. All the enzymes demonstrated proteolytic activity(data not shown).

Once the enzymes were verified to be functional they were incubated at37° C. with the various RAGE-Fc fusion proteins for up to 24 hours. Inbrief, 0.06 μM of ADAM10 (Specific Activity: 1 μg of ADAM10 cleaves 20pmol/min/μg of substrate; 50,000 μg=1 Unit), 0.01 μM of MMP9 (SpecificActivity: 1 μg of MMP9 cleaves 1,300 pmol/min/μg of substrate; 769 μg=1Unit), or 0.002 μM of trypsin (Specific Activity: 1 μg of Trypsincleaves 2,500 pmol/min/μg of substrate; 400 μg=1 Unit) were incubatedwith 2.5 μM of RAGE-Fc fusion protein. The enzymatic reaction wasstopped by adding an anionic detergent 1% lithium dodecyl sulfate (LDS),at the following time points: 0, 2, 15, 24 hours. As a control, theRAGE-Fc fusion proteins were incubated without enzyme to ensure thatthey were stable over the 24-hour time course of the experiment. Thesamples were then run on SDS-PAGE using SYPRO Ruby protein gel stain.Each sample was run under reducing (0.1 M DTT) conditions. The gels wereimaged on Bio-Rad Molecular Imager and the bands were analyzed usingImage Lab Software.

Results of the proteolytic stability experiments are shown in FIGS.7A-7G, FIGS. 8A-8D, and Table 5. The results show that Constructs #9(RAGE-Fc fusion lacking the C-terminal 13 amino acid RAGE stem), and 10,12, and 16 (esRAGE-Fc fusions) were more resistant to proteolyticcleavage by MMP9 and trypsin and Constructs #12 and 16 were moreresistant to proteolytic cleavage by ADAM10, as compared to Construct #1(commercial RAGE-Fc fusion protein without the additional 16 amino acidsat the carboxy terminus of the RAGE polypeptide) (SEQ ID NO: 5). Allprotease experiments were conducted under non-reducing conditions topreserve disulfide bonds in the Fc polypeptide during the stability timecourse. Reaction products were run on SDS-PAGE under reducing conditionsin order to observe the reduced monomeric products (FIGS. 7A-7G).Examples of quantification data of the SDS-PAGE results at a specifictime point are shown in Table 5. Data is presented as percent offull-length RAGE-Fc fusion protein (FL) remaining after the indicatedtreatment. The full-length proteins were quantified by fluorescent imageintensities on the SDS-PAGE gel. Percentages are expressed as offunction of the time zero band intensity for each condition. FIGS. 8A-8Dshow time course proteolysis data for the fusion proteins. Data shown isquantified from fluorescent bands of SDS-PAGE gels run under reducingconditions. Percent change is expressed as percent of the full lengthRAGE-Fc construct present at the indicated time point. Table 6identifies the SEQ ID NO. of each construct tested.

TABLE 5 Full Length Construct Protease Time (h) #1 #9 #10 #12 #16 ADAM1015 84% 91% 92% 100% 100% MMP9 15 15% 52% 38%  51%  65% Trypsin 15  0%39% 39%  35%  34%

TABLE 6 RAGE Fc Construct SEQ ID NO #1 5 #9 53 #10 12 #12 15 #16 16

Example 4: Assessing Susceptability to Degradation in Serum.

The RAGE-Fc fusion proteins were assessed for their vulnerability tocleavage by enzymes found in normal human serum. For comparison, theesRAGE-Fc fusion proteins of the present disclosure were tested againsta purified version identical to commercially available RAGE-Fcconstruct.

The serum was verified to contain active enzymes under set assayconditions by demonstrating cleavage of a fluorogenic peptide substrate.In brief, the serum was incubated with 10 μM of fluorogenic peptidesubstrate [Mca-KPLGL-Dpa-AR-NH2 (SEQ ID NO: 75)]. The fluorescence wasmeasured kinetically at 320 nm excitation and 405 nm emission via anautomated fluorescence microplate reader. The serum demonstratedproteolytic activity (data not shown).

Once the serum was verified to contain active enzymes it was incubatedat 37° C. with the various RAGE-Fc fusion proteins for up to 138 hours.In brief, 75% (v/v) of serum was incubated with 25% (v/v) of 2 μM ofRAGE-Fc fusion protein in PBS. The enzymatic reaction was stopped byadding an anionic detergent 1% lithium dodecyl sulfate (LDS), at thefollowing time points: 0, 17, 49, 138 hours. As a control, the serum wastested without RAGE-Fc fusion protein to ensure no endogenous solubleRAGE was detected in the serum. The serum samples were tested withWestern Blot to detect the presence of the constructs. In brief, thesamples were run on SDS-PAGE under reducing conditions (0.1 M DTT), thentransferred to PVDF membrane and stained with Ponceau to ensure thetransfer was successful. The PVDF membrane was then blocked with 5% BSAin TBS-Tween for 1 hour at room temperature, then incubated with theprimary antibody diluted 1:500 in TBS-Tween containing 5% BSA(Invitrogen, Cat. No. 701316) overnight at 4° C. The membrane was thenwashed five times with TBS-Tween for 5 min per wash and then incubatedwith the secondary antibody diluted 1:5000 in TBS-Tween containing 5%BSA (GenTex, Cat No. GTX213110-01) for 1 hour at room temperature. Themembrane was again washed five times with TBS-Tween for 5 min per wash,and then detected using (ECL) chemiluminescence. The gels were imaged onBio-Rad Molecular Imager and the bands were analyzed using Image LabSoftware.

Results of the serum stability experiments are shown in FIGS. 9A-9D,FIG. 10, and Table 7. The results show that Constructs #9, 12, and 16were more resistant to proteolytic cleavage by enzymes found in serum ascompared to Constructs #1 and #10. All serum stability experiments wereconducted under non-reducing conditions to preserve disulfide bonds inthe Fc polypeptide during the stability time course. Reaction productswere run on SDS-PAGE under reducing conditions in order to observe thereduced monomeric products as seen on the Western Blots (FIGS. 9A-9D).Quantification data of the Western Blot results are shown in Table 7.Data is presented as percent of full-length RAGE-Fc fusion protein (FL)remaining after the indicated time point. The full-length proteins werequantified by image intensities on the Western Blot membrane.Percentages are expressed as of function of the time zero band intensityfor each condition. FIGS. 8A-8D show time course proteolysis data forthe fusion proteins. Data shown is quantified from intensity bands ofthe Western Blot membranes run under reducing conditions. Percent changeis expressed as percent of the full length RAGE-Fc construct present atthe indicated time point. Table 4 identifies the SEQ ID NO. of eachconstruct tested.

TABLE 7 Full Length Construct Time (h) #1 #9 #10 #12 #16 0 100% 100%100% 100% 100% 17 65% 100% 100% 99% 81% 49 47% 100% 74% 86% 64% 138 0%22% 0% 23% 17%

Example 5: Assessing Thermal Stability and Aggregation

Dynamic light scattering (DLS) was used to analyze the aggregationtemperature (T_(agg)) of RAGE-Fc fusion proteins in the same buffersolution. DLS was performed using the DynaPro® NanoStar® instrument tomeasure the effect of temperature on translational diffusioncoefficients (D_(t)) of nanoparticles and colloids in solution byquantifying dynamic fluctuations in scattered light. Sizes and sizedistributions, in turn, are calculated from the diffusion coefficientsin terms of hydrodynamic diameter (d_(h)). Results are shown in FIGS.11A-11D: Construct #1 (FIG. 11A); Construct #10 (FIG. 11B); Construct#12 (FIG. 11C); Construct #16 (FIG. 11D). DLS profiles of fusionproteins were analyzed by the framework of Onset model, the dotsindicate the raw data while the green solid line indicates the fittingcurve by the model. The results show that Constructs #10 and #12(esRAGE-Fc fusions) have enhanced thermal stability as compared toConstruct #1. Results from this analysis including hydrodynamic radius(nm) and T_(agg) (° C.) are shown in Table 8.

TABLE 8 Concentration Construct (mg/ml) T_(agg) (° C.) Radius (nm) #12.26 61.39 7.49 #10 2.86 63.40 8.06 #12 1.55 67.24 7.50 #16 2.00 52.948.68

Example 6: Improved Manufacturability

Further modified RAGE-Fc fusion proteins were constructed to test forimprovement of protein expression and manufacturability of the fusionprotein. Improved manufacturability manifests in one or more of thefollowing ways: higher expression, increased stability, or improvedsolubility. Solubility may be assessed by SDS-PAGE under reducing andnon-reducing conditions, followed by Western blot. In contrast to theprior art, the improved molecules of the present disclosure demonstratereduced tendency to aggregate as shown by distinct protein bands visibleunder reducing conditions compared to smeared bands visible undernon-reducing conditions (see FIGS. 2A-2L, FIGS. 3A-3J, FIGS. 4A-4I, andFIGS. 5A-5F, comparing bands in lane 1 (reducing condition) with bandsin lane 2 (non-reducing conditions)).

For example, esRAGE-Fc fusion proteins were constructed using at least aportion of the hinge region of alternative human IgG polypeptides as alinker between the C-terminus of esRAGE and the amino terminus of the Fcpolypeptide of the fusion protein. A RAGE-Fc fusion protein was alsoconstructed using a RAGE polypeptide with a shortened stem regionlacking the C-terminal 13 amino acid residues, with a portion of thehinge region of alternative human IgG polypeptides as a linker betweenthe C-terminus of RAGE and the amino terminus of the Fc polypeptide ofthe fusion protein. Additional modified fusion proteins were generatedby introducing amino acid substitutions into the esRAGE polypeptide,and/or the Fc polypeptide of the fusion protein. Fusion proteinscomprising alternative linkers and amino acid substitutions weregenerated using overlap PCR mutagenesis according to known methods.

Testing of esRAGE-Fc fusion proteins comprising linkers from alternativeIgG hinge regions and esRAGE-Fc fusion proteins comprising amino acidsubstitutions was performed as follows. Polynucleotides encodingesRAGE-Fc fusion proteins comprising an IgG4 hinge linker (SEQ ID NO:39), a RAGE polypeptide with a shortened stem region lacking theC-terminal 13-amino acid residues (SEQ ID NO: 54), or polynucleotidesencoding fusion proteins comprising an IgG2 linker (SEQ ID NO: 41) wereexpressed in CHO-3E7 cells as described in Example 1. Further,polynucleotides encoding esRAGE-Fc fusion proteins comprising aminosubstitutions M252Y, S254T, and T256E in the Fc polypeptide (SEQ ID NO:44) were also expressed in CHO-3E7 cells as described in Example 1. Thecultures were grown for six days following transfection; on day 6 thecell culture supernatant was collected and used for purification asdescribed in Example 1. Purified protein was analyzed by SDS-PAGE underreducing and non-reducing conditions and by Western blot using a primaryGoat Anti-Human IgG-HRP antibody (GenScript, Cat. No. A00166). Proteinconcentration was determined by Bradford assay using BSA as a proteinstandard. Tables 5 and 6 show the concentration, purity, and totalpurified protein yield for each fusion protein.

The esRAGE-Fc fusion protein encoded by the amino acid sequence setforth in SEQ ID NO: 12 (nucleotide sequence set forth in SEQ ID NO: 39)differs from the fusion protein encoded by the amino acid sequence setforth in SEQ ID NO 15 (nucleotide sequence set forth in SEQ ID NO: 41)only by the IgG hinge from which the linker is derived. Further, theesRAGE-Fc fusion protein encoded by the amino acid sequence set forth inSEQ ID NO: 16 (nucleotide sequence set forth in SEQ ID NO: 43) differsfrom the fusion protein encoded by the amino acid sequence set forth inSEQ ID NO: 15 (nucleotide sequence set forth in SEQ ID NO: 41) only bythe amino acid substitutions at positions 252, 254, and 256 (EUnumbering) of the Fc polypeptide. The results shown in Table 9demonstrate that the purity and yield, and thus the manufacturability ofthe fusion protein may be improved by replacing a linker from the IgG4hinge with a linker from the IgG2 hinge. Similarly, the results shown inTable 9 demonstrate that manufacturability of the fusion protein isimproved by incorporating amino acid substitutions M252Y, S254T, andT256E (EU numbering) in the Fc polypeptide of the fusion protein.

TABLE 9 Amino acid Nucleotide Total sequence sequence Construct nameConcentration Purity protein SEQ ID NO: 12 SEQ ID NO: 39 Construct #10:RAGEV-C1-C2-V1 stem 0.29 mg/mL 70% 580 μg(M/A)-IgG4-hinge(S/P-AA)-(IgG4CH2—CH3) SEQ ID NO: 14 SEQ ID NO: 40Construct #11: RAGEV-C1-C2-V1 stem 0.16 mg/mL 80% 640 μg(M/A)-VH8aa-IgG4-hinge(S/P-AA)- (IgG4CH2—CH3) SEQ ID NO: 15 SEQ ID NO:41 Construct #12: RAGEV-C1-C2- 0.19 mg/mL 80% 760 μgV1stem(M/A)-IgG2lowerhinge- (IgG4CH2—CH3) SEQ ID NO: 16 SEQ ID NO: 43Construct #16: RAGE V-C1-C2- 0.21 mg/mL 90% 1.68 mg V1stem(M/A)-IgG2lower hinge- (IgG4CH2—CH3)-YTE

Data showing the concentration, purity, and total purified protein yieldfor RAGE-Fc fusion proteins expressed at 1L scale and purified usingMonofinity A Resin affinity purification, followed by HiLoad26/600Superdex200 pg size exclusion chromatography. The results shown in Table10 demonstrate that the purity and yield, and thus the manufacturabilityof the fusion protein in scaled-up production may be improved byreplacing a linker from the IgG4 hinge with a linker from the IgG2hinge. Similarly, the results shown in Table 10 demonstrate thatmanufacturability of the fusion protein is improved by incorporatingamino acid substitutions M252Y, S254T, and T256E (EU numbering) in theFc polypeptide of the fusion protein.

TABLE 10 Amino acid Nucleotide Total sequence sequence Construct nameConcentration Purity protein SEQ ID NO: 5 SEQ ED NO: 38 Construct #1:RAGE V-C1-C2-Natural 0.58 mg/mL 90% 18.27 mg  Stem-short linker-IgG1hinge- (IgG1CH2—CH3) SEQ ID NO: 54 SEQ ID NO: 57 Construct #9: RAGEV-C1-C2- 0.48 mg/mL 90% 2.88 mg shortenedstem-VH8aa-IgG4-hinge(S/P-AA)—(IgG4CH2—CH3) SEQ ID NO: 12 SEQ ID NO: 39 Construct #10:RAGEV-C1-C2-V1 stem 0.38 mg/mL 90% 4.94 mg(M/A)-IgG4-hinge(S/P-AA)-(IgG4CH2—CH3) SEQ ID NO: 14 SEQ ID NO: 40Construct #11: RAGEV-C1-C2-V1 stem 0.43 mg/mL 90% 4.30 mg(M/A)-VH8aa-IgG4-hinge(S/P-AA)- (IgG4CH2—CH3) SEQ ID NO: 15 SEQ ID NO:41 Construct #12: RAGEV-C1-C2- 0.66 mg/mL 90% 7.26 mgV1stem(M/A)-IgG2lowerhinge- (IgG4CH2—CH3) SEQ ID NO: 16 SEQ ID NO: 43Construct #16: RAGE V-C1-C2- 0.43 mg/mL 90% 12.25 mg  V1stem(M/A)-IgG2lower hinge- (IgG4CH2—CH3)-YTE

Data showing the concentration, purity, and total purified protein yieldfor additional RAGE-Fc fusion proteins is provided in Table 11.

TABLE 11 Amino acid Nucleotide Total sequence sequence Construct nameConcentration Purity protein SEQ ID NO: 5 SEQ ID NO: 38 Construct #1:RAGE V-C1-C2-Natural 0.26 mg/mL 80% 1.56 mg Stem-short linker-IgG1hinge-(IgG1CH2—CH3) SEQ ID NO: 37 SEQ ID NO: 42 Construct #13:RAGEV-C1-C2 0.15 mg/mL 65% 450 μg (N25E/G82S)-Natural Stem-short linker-IgG1 hinge-(IgG1CH2—CH3) SEQ ID NO: 17 SEQ ID NO: 44 Construct #17: RAGEV-C1-C2- 0.21 mg/mL 80% 2.10 mg V1stem(M/A)-(N25E)-IgG2 lower hinge-(IgG4CH2—CH3) SEQ ID NO: 18 SEQ ID NO: 45 Construct #18: RAGE V-C1-C2-0.14 mg/mL 75% 1.40 mg V1stem(M/A)-(N25Q)-IgG2 lower hinge-(IgG4CH2—CH3) SEQ ID NO: 19 SEQ ID NO: 46 Construct #19: RAGE V-C1-C2-0.13 mg/mL 85% 780 μg V1stem(M/A)-(G82S)-IgG2 lower hinge- (IgG4CH2—CH3)SEQ ID NO: 20 SEQ ID NO: 47 Construct #20: RAGE V-C1-C2- 0.10 mg/mL 70%1.05 mg V1stem(M/A)-(N25E/G82S)-IgG2 lower hinge-(IgG4CH2—CH3) SEQ IDNO: 21 SEQ ID NO: 48 Construct #21: RAGE V-C1-C2- 0.12 mg/mL 75% 1.08 mgV1stem(M/A)-(N25Q/G82S)-IgG2 lower hinge-(IgG4CH2—CH3) SEQ ID NO: 22 SEQID NO: 49 Construct #22: RAGE V-C1-C2- 0.13 mg/mL N/A 780 μgV1stem(M/A)-(N81A)-IgG2 lower hinge- (IgG4CH2—CH3) SEQ ID NO: 23 SEQ IDNO: 50 Construct #23: RAGE V-C1-C2-  52 μg/mL 35% 676 μgV1stem(M/A)-(N25E/N81A)-IgG2 lower hinge—(IgG4CH2—CH3) SEQ ID NO: 24 SEQID NO: 51 Construct #24: RAGE V-C1-C2- 0.13 mg/mL N/A 1.69 mgV1stem(M/A)-(N25Q/N81A)-IgG2 lower hinge—(IgG4CH2—CH3)

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

INFORMAL SEQUENCE LISTING SEQ ID NO DESCRIPTION SEQUENCE SEQ ID NO: 1esRAGE including the MAAGTAVGAW VLVLSLWGAV VGAQNITARInatural leader sequence GEPLVLKCKG APKKPPQRLE WKLNTGRTEA(natural leader sequence is WKVLSPQGGG PWDSVARVLP NGSLFLPAVG underlined)IQDEGIFRCQ AMNRNGKETK SNYRVRVYQI PGKPEIVDSA SELTAGVPNK VGTCVSEGSYPAGTLSWHLD GKPLVPNEKG VSVKEQTRRH PETGLFTLQS ELMVTPARGG DPRPTFSCSFSPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCE VPAQPSPQIHWMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISI IEPGEEGPTAGEGFDKVREA EDSPQHM SEQ ID NO: 2 15 of 16 AA of C-term. EGFDKVREA EDSPQHsequence unique to esRAGE SEQ ID NO: 3 AA sequence of hRAGE-MAAGTAVGAW VLVLSLWGAV VGAQNITARI IgG4Fc fusion protein of USGEPLVLKCKG APKKPPQRLE WKLNTGRTEA 9,399,668-SEQ ID NO: 6 inWKVLSPQGGG PWDSVARVLP NGSLFLPAVG the ′668 patent (includes theTQDEGIFRCQ AMNRNGKETK SNYRVRVYQI natural leader sequence,PGKPEIVDSA SELTAGVPNK VGTCVSEGSY underlined)PAGTLSWHLD GKPLVPNEKG VSVKEQTRRH PETGLFTLQS ELMVTPARGG DPRPTFSCSPSPGLPRRRAL HTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCE VPAQPSPQIHWMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISI IEPGEEGPTAGSVGGSGLGT LALAASTKGP SVFPLAPCSR STSESTAALG CLVKDYFPEP VTVSWNSGALTSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTKTYTCNVD HKPSNTKVDK RVESKYGPPCPSCPAPEFLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SQEDPEVQFN WYVDGVEVHNAKTKPREEQF NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK GLPSSTEKTI SKAKGQPREPQVYTLPPSQE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFLYSRLTVDKSR WQEGNVFSCS VMHEALHNHY TQKSLSLSLG K SEQ ID NO: 4Sequence corresponding to xYxTxE M252Y/S254T/T256Emutation in CH2 domain of IgG Fc SEQ ID NO: 5 Sequence corresponding toAQNITARIGE PLVLKCKGAP KKPPQRLE Construct #1 (matureWKLNTGRTEA WKVLSPQGGG PWDSVARVLP protein; lacking the naturalNGSLFLPAVG IQDEGIFRCQ AMNRNGKETK leader sequence)SNYRVRVYQI PGKPEIVDSA SELTAGVPNK VGTCVSEGSY PAGTLSWHLD GKPLVPNEKGVSVKEQTRRH PETGLFTLQS ELMVTPARGG DPRPTFSCSF SPGLPRHRAL RTAPIQPRVWEPVPLEEVQL VVEPEGGAVA PGGTVTLTCE VPAQPSPQIH WMKDGVPLPL PPSPVLILPEIGPQDQGTYS CVATHSSHGP QESRAVSISI IEPGEEGPTA GSVGGSGLGT LALAIEGRMPKSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNWYVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK SEQ ID NO: 6Sequence of extracellular AQNITARIGE PLVLKCKGAP KKPPQRLEdomain of WT hRAGE (not WKLNTGRTEA WKVLSPQGGG PWDSVARVLPthe splice variant) (mature NGSLFLPAVG IQDEGIFRCQ AMNRNGKETKprotein; lacking the natural SNYRVRVYQI PGKPEIVDSA SELTAGVPNKleader sequence) VGTCVSEGSY PAGTLSWHLD GKPLVPNEKGVSVKEQTRRH PETGLFTLQS ELMVTPARGG DPRPTFSCSF SPGLPRHRAL RTAPIQPRVWEPVPLEEVQL VVEPEGGAVA PGGTVTLTCE VPAQPSPQIH WMKDGVPLPL PPSPVLILPEIGPQDQGTYS CVATHSSHGP QESRAVSISI IEPGEEGPTA G SEQ ID NO: 7AA359-590 of construct #17 GG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSQEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGK EYKCKVSNKGLPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGKSEQ ID NO: 8 Modified IgG4 (S/P-AA) ESKYGPPCPPCPAPEAAhinge that is present in esRAGE-Fc linker in constructs #10, 11, 33, 35,and 36 SEQ ID NO: 9 IgG2 lower hinge that is VECPPCAPPVApresent in esRAGE-Fc linker in constructs #12, 16-29, 31- 32SEQ ID NO: 10 IgG2 complete hinge that is ERKCCVECPPCAPPVApresent in esRAGE-Fc linker in construct #30 SEQ ID NO: 11IgG1 hinge that is present in EPKSCDKTHTCPPCPAPEAA esRAGE-Fc linker inconstruct #34 SEQ ID NO: 12 Construct #10 AQNITARIGE PLVLKCKGAP KKPPQRLERAGE V-C1-C2- WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-IgG4-hingeNGSLFLPAVG IQDEGIFRCQ AMNRNGKETK (S/P-AA)-(IgG4CH2-CH3)SNYRVRVYQI PGKPEIVDSA SELTAGVPNK (mature protein; lacking theVGTCVSEGSY PAGTLSWHLD GKPLVPNEKG natural leader sequence)VSVKEQTRRH PETGLFTLQS ELMVTPARGG DPRPTFSCSF SPGLPRHRAL RTAPIQPRVWEPVPLEEVQL VVEPEGGAVA PGGTVTLTCE VPAQPSPQIH WMKDGVPLPL PPSPVLILPEIGPQDQGTYS CVATHSSHGP QESRAVSISI IEPGEEGPTA GEGFDKVREA EDSPQHAESKYGPPCPPCPA PEAAGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSQEDP EVQFNWYVDGVEVHNAKTKP REEQFNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSS IEKTISKAKGQPREPQVYTL PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK SEQ ID NO: 13Shortened stem-VH8aa- GTLVTVSS IgG4-hinge(S/P-AA) SEQ ID NO: 14Construct #11 AQNITARIGE PLVLKCKGAP KKPPQRLE RAGE V-C1-C2-WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-VH8aa-IgG4-NGSLFLPAVG IQDEGIFRCQ AMNRNGKETK hinge (S/P-AA)-SNYRVRVYQI PGKPEIVDSA SELTAGVPNK (IgG4CH2-CH3) (matureVGTCVSEGSY PAGTLSWHLD GKPLVPNEKG protein; lacking the naturalVSVKEQTRRH PETGLFTLQS ELMVTPARGG leader sequence)DPRPTFSCSF SPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCEVPAQPSPQIH WMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISIIEPGEEGPTA GEGFDKVREA EDSPQHAGTL VTVSSESKYG PPCPPCPAPE AAGGPSVFLFPPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVVSVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVFSCSVMHEALH NHYTQKSLSL SLGK SEQ ID NO: 15 Construct #12AQNITARIGE PLVLKCKGAP KKPPQRLE RAGE V-C1-C2-WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-IgG2 lowerNGSLFLPAVG IQDEGIFRCQ AMNRNGKETK hinge-(IgG4CH2-CH3)SNYRVRVYQI PGKPEIVDSA SELTAGVPNK (mature protein; lacking theVGTCVSEGSY PAGTLSWHLD GKPLVPNEKG natural leader sequence)VSVKEQTRRH PETGLFTLQS ELMVTPARGG DPRPTFSCSF SPGLPRHRAL RTAPIQPRVWEPVPLEEVQL VVEPEGGAVA PGGTVTLTCE VPAQPSPQIH WMKDGVPLPL PPSPVLILPEIGPQDQGTYS CVATHSSHGP QESRAVSISI IEPGEEGPTA GEGFDKVREA EDSPQHAVECPPCAPPVAGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNAKTKPREEQFN STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQVYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLYSRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGK SEQ ID NO: 16 Construct #16AQNITARIGE PLVLKCKGAP KKPPQRLE (#12 + YTE)WKLNTGRTEA WKVLSPQGGG PWDSVARVLP RAGE V-C1-C2-NGSLFLPAVG IQDEGIFRCQ AMNRNGKETK V1 stem(M/A)-IgG2 lowerSNYRVRVYQI PGKPEIVDSA SELTAGVPNK hinge-(IgG4CH2-CH3)-VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG YTE (mature protein;VSVKEQTRRH PETGLFTLQS ELMVTPARGG lacking the natural leaderDPRPTFSCSF SPGLPRHRAL RTAPIQPRVW sequence)EPVPLEEVQL VVEPEGGAVA PGGTVTLTCE VPAQPSPQIH WMKDGVPLPL PPSPVLILPEIGPQDQGTYS CVATHSSHGP QESRAVSISI IEPGEEGPTA GEGFDKVREA EDSPQHAVECPPCAPPVAGG PSVFLFPPKP KDTLYITREP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNAKTKPREEQFN STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQVYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLYSRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGK SEQ ID NO: 17 Construct #17AQEITARIGE PLVLKCKGAP KKPPQRLE RAGE V-C1-C2-WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-(N25E)-IgG2NGSLFLPAVG IQDEGIFRCQ AMNRNGKETK lower hinge-(IgG4CH2-SNYRVRVYQI PGKPEIVDSA SELTAGVPNK CH3) (mature protein;VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG lacking the natural leaderVSVKEQTRRH PETGLFTLQS ELMVTPARGG sequence)DPRPTFSCSF SPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCEVPAQPSPQIH WMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISIIEPGEEGPTA GEGFDKVREA EDSPQHAVEC PPCAPPVAGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGKEYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYTQKSLSLSLGK SEQ ID NO: 18 Construct #18 AQQITARIGE PLVLKCKGAP KKPPQRLERAGE V-C1-C2- WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-(N25Q)-IgG2NGSLFLPAVG IQDEGIFRCQ AMNRNGKETK lower hinge-(IgG4CH2-SNYRVRVYQI PGKPEIVDSA SELTAGVPNK CH3) (mature protein;VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG lacking the natural leaderVSVKEQTRRH PETGLFTLQS ELMVTPARGG sequence)DPRPTFSCSF SPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCEVPAQPSPQIH WMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISIIEPGEEGPTA GEGFDKVREA EDSPQHAVEC PPCAPPVAGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGKEYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYTQKSLSLSLGK SEQ ID NO: 19 Construct #19 AQNITARIGE PLVLKCKGAP KKPPQRLERAGE V-C1-C2- WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-(G82S)-IgG2NSSLFLPAVG IQDEGIFRCQ AMNRNGKETK lower hinge-(IgG4CH2-SNYRVRVYQI PGKPEIVDSA SELTAGVPNK CH3) (mature protein;VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG lacking the natural leaderVSVKEQTRRH PETGLFTLQS ELMVTPARGG sequence)DPRPTFSCSF SPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCEVPAQPSPQIH WMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISIIEPGEEGPTA GEGFDKVREA EDSPQHAVEC PPCAPPVAGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGKEYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYTQKSLSLSLGK SEQ ID NO: 20 Construct #20 AQEITARIGE PLVLKCKGAP KKPPQRLERAGE V-C1-C2- WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-NSSLFLPAVG IQDEGIFRCQ AMNRNGKETK (N25E/G82S)-IgG2 lowerSNYRVRVYQI PGKPEIVDSA SELTAGVPNK hinge-(IgG4CH2-CH3)VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG (mature protein; lacking theVSVKEQTRRH PETGLFTLQS ELMVTPARGG natural leader sequence)DPRPTFSCSF SPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCEVPAQPSPQIH WMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISIIEPGEEGPTA GEGFDKVREA EDSPQHAVEC PPCAPPVAGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGKEYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYTQKSLSLSLGK SEQ ID NO: 21 Construct #21 AQQITARIGE PLVLKCKGAP KKPPQRLERAGE V-C1-C2- WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-NSSLFLPAVG IQDEGIFRCQ AMNRNGKETK (N25Q/G82S)-IgG2 lowerSNYRVRVYQI PGKPEIVDSA SELTAGVPNK hinge-(IgG4CH2-CH3)VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG (mature protein; lacking theVSVKEQTRRH PETGLFTLQS ELMVTPARGG natural leader sequence)DPRPTFSCSF SPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCEVPAQPSPQIH WMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISIIEPGEEGPTA GEGFDKVREA EDSPQHAVEC PPCAPPVAGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGKEYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYTQKSLSLSLGK SEQ ID NO: 22 Construct #22 AQNITARIGE PLVLKCKGAP KKPPQRLERAGE V-C1-C2- WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-(N81A)-IgG2AGSLFLPAVG IQDEGIFRCQ AMNRNGKETK lower hinge-(IgG4CH2-SNYRVRVYQI PGKPEIVDSA SELTAGVPNK CH3) (mature protein;VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG lacking the natural leaderVSVKEQTRRH PETGLFTLQS ELMVTPARGG sequence)DPRPTFSCSF SPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCEVPAQPSPQIH WMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISIIEPGEEGPTA GEGFDKVREA EDSPQHAVEC PPCAPPVAGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGKEYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYTQKSLSLSLGK SEQ ID NO: 23 Construct #23 AQEITARIGE PLVLKCKGAP KKPPQRLERAGE V-C1-C2- WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-AGSLFLPAVG IQDEGIFRCQ AMNRNGKETK (N25E/N81A)-IgG2 lowerSNYRVRVYQI PGKPEIVDSA SELTAGVPNK hinge-(IgG4CH2-CH3)VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG (mature protein; lacking theVSVKEQTRRH PETGLFTLQS ELMVTPARGG natural leader sequence)DPRPTFSCSF SPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCEVPAQPSPQIH WMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISIIEPGEEGPTA GEGFDKVREA EDSPQHAVEC PPCAPPVAGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGKEYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYTQKSLSLSLGK SEQ ID NO: 24 Construct #24 AQQITARIGE PLVLKCKGAP KKPPQRLERAGE V-C1-C2- WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-AGSLFLPAVG IQDEGIFRCQ AMNRNGKETK (N25Q/N81A)-IgG2 lowerSNYRVRVYQI PGKPEIVDSA SELTAGVPNK hinge-(IgG4CH2-CH3)VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG (mature protein; lacking theVSVKEQTRRH PETGLFTLQS ELMVTPARGG natural leader sequence)DPRPTFSCSF SPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCEVPAQPSPQIH WMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISIIEPGEEGPTA GEGFDKVREA EDSPQHAVEC PPCAPPVAGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGKEYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYTQKSLSLSLGK SEQ ID NO: 25 Construct #25 AQEITARIGE PLVLKCKGAP KKPPQRLERAGE V-C1-C2- WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-(N25E)-IgG2NGSLFLPAVG IQDEGIFRCQ AMNRNGKETK lower hinge-(IgG4CH2-SNYRVRVYQI PGKPEIVDSA SELTAGVPNK CH3)-YTE (mature protein;VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG lacking the natural leaderVSVKEQTRRH PETGLFTLQS ELMVTPARGG sequence)DPRPTFSCSF SPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCEVPAQPSPQIH WMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISIIEPGEEGPTA GEGFDKVREA EDSPQHAVEC PPCAPPVAGG PSVFLFPPKP KDTLYITREPEVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGKEYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYTQKSLSLSLGK SEQ ID NO: 26 Construct #26 AQQITARIGE PLVLKCKGAP KKPPQRLERAGE V-C1-C2- WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-(N25Q)-IgG2NGSLFLPAVG IQDEGIFRCQ AMNRNGKETK lower hinge-(IgG4CH2-SNYRVRVYQI PGKPEIVDSA SELTAGVPNK CH3)-YTE (mature protein;VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG lacking the natural leaderVSVKEQTRRH PETGLFTLQS ELMVTPARGG sequence)DPRPTFSCSF SPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCEVPAQPSPQIH WMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISIIEPGEEGPTA GEGFDKVREA EDSPQHAVEC PPCAPPVAGG PSVFLFPPKP KDTLYITREPEVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGKEYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYTQKSLSLSLGK SEQ ID NO: 27 Construct #27 AQNITARIGE PLVLKCKGAP KKPPQRLERAGE V-C1-C2- WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-(G82S)-IgG2NSSLFLPAVG IQDEGIFRCQ AMNRNGKETK lower hinge-(IgG4CH2-SNYRVRVYQI PGKPEIVDSA SELTAGVPNK CH3)-YTE (mature protein;VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG lacking the natural leaderVSVKEQTRRH PETGLFTLQS ELMVTPARGG sequence)DPRPTFSCSF SPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCEVPAQPSPQIH WMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISIIEPGEEGPTA GEGFDKVREA EDSPQHAVEC PPCAPPVAGG PSVFLFPPKP KDTLYITREPEVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGKEYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYTQKSLSLSLGK SEQ ID NO: 28 Construct #28 AQEITARIGE PLVLKCKGAP KKPPQRLERAGE V-C1-C2- WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-NSSLFLPAVG IQDEGIFRCQ AMNRNGKETK (N25E/G82S)-IgG2 lowerSNYRVRVYQI PGKPEIVDSA SELTAGVPNK hinge-(IgG4CH2-CH3)-VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG YTE (mature protein;VSVKEQTRRH PETGLFTLQS ELMVTPARGG lacking the natural leadersDPRPTFSCSF SPGLPRHRAL RTAPIQPRVW sequence)EPVPLEEVQL VVEPEGGAVA PGGTVTLTCE VPAQPSPQIH WMKDGVPLPL PPSPVLILPEIGPQDQGTYS CVATHSSHGP QESRAVSISI IEPGEEGPTA GEGFDKVREA EDSPQHAVECPPCAPPVAGG PSVFLFPPKP KDTLYITREP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNAKTKPREEQFN STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQVYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLYSRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGK SEQ ID NO: 29 Construct #29AQQITARIGE PLVLKCKGAP KKPPQRLE RAGE V-C1-C2-WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-NSSLFLPAVG IQDEGIFRCQ AMNRNGKETK (N25Q/G82S)-IgG2 lowerSNYRVRVYQI PGKPEIVDSA SELTAGVPNK hinge-(IgG4CH2-CH3)-VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG YTE (mature protein;VSVKEQTRRH PETGLFTLQS ELMVTPARGG lacking the natural leadersDPRPTFSCSF SPGLPRHRAL RTAPIQPRVW sequence)EPVPLEEVQL VVEPEGGAVA PGGTVTLTCE VPAQPSPQIH WMKDGVPLPL PPSPVLILPEIGPQDQGTYS CVATHSSHGP QESRAVSISI IEPGEEGPTA GEGFDKVREA EDSPQHAVECPPCAPPVAGG PSVFLFPPKP KDTLYITREP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNAKTKPREEQFN STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQVYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLYSRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGK SEQ ID NO: 30 Construct #30AQNITARIGE PLVLKCKGAP KKPPQRLE RAGE V-C1-C2-WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-IgG2NGSLFLPAVG IQDEGIFRCQ AMNRNGKETK complete hinge-(IgG4CH2-SNYRVRVYQI PGKPEIVDSA SELTAGVPNK CH3)-YTE (mature protein;VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG lacking the natural leaderVSVKEQTRRH PETGLFTLQS ELMVTPARGG sequence)DPRPTFSCSF SPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCEVPAQPSPQIH WMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISIIEPGEEGPTA GEGFDKVREA EDSPQHAERK CCVECPPCAP PVAGGPSVFL FPPKPKDTLYITREPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRV VSVLTVLHQDWLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLP PSQEEMTKNQ VSLTCLVKGFYPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEALHNHYTQKSLS LSLGK SEQ ID NO: 31 Construct #31AQNITARIGE PLVLKCKGAP KKPPQRLE RAGE V-C1-C2-WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-IgG2 lowerNGSLFLPAVG IQDEGIFRCQ AMNRNGKETK hinge-(IgG2CH2-CH3)-SNYRVRVYQI PGKPEIVDSA SELTAGVPNK YTE (mature protein;VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG lacking the natural leaderVSVKEQTRRH PETGLFTLQS ELMVTPARGG sequence)DPRPTFSCSF SPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCEVPAQPSPQIH WMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISIIEPGEEGPTA GEGFDKVREA EDSPQHAVEC PPCAPPVAGP SVFLFPPKPK DTLYITREPEVTCVVVDVSH EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TFRVVSVLTV VHQDWLNGKEYKCKVSNKGL PAPIEKTISK TKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIAVEWESNGQPE NNYKTTPPML DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQKSLSLSPGK SEQ ID NO: 32 Construct #32 AQNITARIGE PLVLKCKGAP KKPPQRLERAGE V-C1-C2- WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-IgG2 lowerNGSLFLPAVG IQDEGIFRCQ AMNRNGKETK hinge-(IgG1CH2-CH3)-SNYRVRVYQI PGKPEIVDSA SELTAGVPNK YTE (mature protein;VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG lacking the natural leaderVSVKEQTRRH PETGLFTLQS ELMVTPARGG sequence)DPRPTFSCSF SPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCEVPAQPSPQIH WMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISIIEPGEEGPTA GEGFDKVREA EDSPQHAVEC PPCAPPVAGG PSVFLFPPKP KDTLYITREPEVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYTQKSLSLSPGK SEQ ID NO: 33 Construct #33 AQNITARIGE PLVLKCKGAP KKPPQRLERAGE V-C1-C2- WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-IgG4-hingeNGSLFLPAVG IQDEGIFRCQ AMNRNGKETK (S/P-AA)-(IgG1CH2-SNYRVRVYQI PGKPEIVDSA SELTAGVPNK CH3)-YTE (mature protein;VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG lacking the natural leadersVSVKEQTRRH PETGLFTLQS ELMVTPARGG sequence)DPRPTFSCSF SPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCEVPAQPSPQIH WMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISIIEPGEEGPTA GEGFDKVREA EDSPQHAESK YGPPCPPCPA PEAAGGPSVF LFPPKPKDTLYITREPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQDWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKGFYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEALHNHYTQKSL SLSPGK SEQ ID NO: 34 Construct #34AQNITARIGE PLVLKCKGAP KKPPQRLE RAGE V-C1-C2-WKLNTGRTEA WKVLSPQGGG PWDSVARVLP V1 stem(M/A)-IgG1 hingeNGSLFLPAVG IQDEGIFRCQ AMNRNGKETK (AA)-(IgG1CH2-CH3)-SNYRVRVYQI PGKPEIVDSA SELTAGVPNK YTE (mature protein;VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG lacking the natural leadersVSVKEQTRRH PETGLFTLQS ELMVTPARGG sequence)DPRPTFSCSF SPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCEVPAQPSPQIH WMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISIIEPGEEGPTA GEGFDKVREA EDSPQHAEPK SCDKTHTCPP CPAPEAAGGP SVFLFPPKPKDTLYITREPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTVLHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCLVKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQ ID NO: 35 Construct #35AQNITARIGE PLVLKCKGAP KKPPQRLE (#10 + YTE)WKLNTGRTEA WKVLSPQGGG PWDSVARVLP RAGE V-C1-C2-RAGE V-NGSLFLPAVG IQDEGIFRCQ AMNRNGKETK C1-C2-V1stem(M/A)-IgG4-SNYRVRVYQI PGKPEIVDSA SELTAGVPNK hinge (S/P-AA)-VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG (IgG4CH2-CH3)-YTEVSVKEQTRRH PETGLFTLQS ELMVTPARGG (mature protein; lacking theDPRPTFSCSF SPGLPRHRAL RTAPIQPRVW natural leaders sequence)EPVPLEEVQL VVEPEGGAVA PGGTVTLTCE VPAQPSPQIH WMKDGVPLPL PPSPVLILPEIGPQDQGTYS CVATHSSHGP QESRAVSISI IEPGEEGPTA GEGFDKVREA EDSPQHAESKYGPPCPPCPA PEAAGGPSVF LFPPKPKDTL YITREPEVTC VVVDVSQEDP EVQFNWYVDGVEVHNAKTKP REEQFNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSS IEKTISKAKGQPREPQVYTL PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK SEQ ID NO: 36Construct #36 AQNITARIGE PLVLKCKGAP KKPPQRLE (#11 + YTE)WKLNTGRTEA WKVLSPQGGG PWDSVARVLP RAGE V-C1-C2-NGSLFLPAVG IQDEGIFRCQ AMNRNGKETK V1 stem(M/A)-VH8aa-IgG4-SNYRVRVYQI PGKPEIVDSA SELTAGVPNK hinge (S/P-AA)-VGTCVSEGSY PAGTLSWHLD GKPLVPNEKG (IgG4CH2-CH3)-YTEVSVKEQTRRH PETGLFTLQS ELMVTPARGG (mature protein; lacking theDPRPTFSCSF SPGLPRHRAL RTAPIQPRVW natural leaders sequence)EPVPLEEVQL VVEPEGGAVA PGGTVTLTCE VPAQPSPQIH WMKDGVPLPL PPSPVLILPEIGPQDQGTYS CVATHSSHGP QESRAVSISI IEPGEEGPTA GEGFDKVREA EDSPQHAGTLVTVSSESKYG PPCPPCPAPE AAGGPSVFLF PPKPKDTLYI TREPEVTCVV VDVSQEDPEVQFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIEKTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKTTPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGKSEQ ID NO: 37 Construct #13 AQEITARIGE PLVLKCKGAP KKPPQRLEWKRAGEV-C1-C2 (N25E/G82S)- LNTGRTEAWK VLSPQGGGPW DSVARVLPNSNatural Stem-short linker-IgG1 TCVSEGSYPA GTLSWHLDGK PLVPNEKGVShinge-(IgG1CH2-CH3) VKEQTRRHPE TGLFTLQSEL MVTPARGGDP(mature protein; lacking the RPTFSCSFSP GLPRHRALRT APIQPRVWEPnatural leaders sequence) VPLEEVQLVV EPEGGAVAPG GTVTLTCEVPAQPSPQIHWM KDGVPLPLPP SPVLILPEIG PQDQGTYSCV ATHSSHGPQE SRAVSISIIEPGEEGPTAGS VGGSGLGTLA LAIEGRMPKS CDKTHTCPPC PAPELLGGPS VFLFPPKPKDTLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVLHQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLVKGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMHEALHNHYTQK SLSLSPGK SEQ ID NO: 38 Nucleotide sequence ofGCTCAGAATATCACCGCCAGAATCGGCGAGCCCCTGGTG Construct #1CTGAAATGTAAAGGCGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGATCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAGAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTCTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGAACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGCCCTACAGCTGGTTCTGTTGGAGGCTCTGGACTGGGCACACTGGCCCTGGCTATTGAGGGCAGAATGCCCAAGTCCTGCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTGCTCGGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCTCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCTTGCCACCTTCTCGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCCTGGCAAATGA SEQ ID NO: 39Nucleotide sequence of GCTCAGAATATCACCGCCAGAATCGGCGAGCCCCTGGTGConstruct #10 CTGAAATGTAAAGGCGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGATCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAGAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTCTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGAACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGAGGCCGAGGATTCTCCTCAGCATGCTGAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAG TCCCTGTCTCTGTCCCTGGGCAAATGASEQ ID NO: 40 Nucleotide sequence ofGCTCAGAATATCACCGCCAGAATCGGCGAGCCCCTGGTG Construct #11CTGAAATGTAAAGGCGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGATCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAGAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTCTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGAACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGAGGCTGAGGACTCTCCTCAGCATGCCGGAACACTGGTCACCGTGTCCTCCGAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCC CTGGGCAAATGA SEQ ID NO: 41Nucleotide sequence of GCTCAGAATATCACCGCCAGAATCGGCGAGCCCCTGGTGConstruct #12 CTGAAATGTAAAGGCGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGATCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAGAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTCTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGAACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGAGGCTGAGGACTCTCCTCAGCATGCCGTGGAATGCCCTCCTTGTGCTCCTCCTGTGGCTGGCGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTG GGCAAATGA SEQ ID NO: 42Nucleotide sequence of GCTCAGGAGATCACCGCCAGAATCGGCGAGCCCCTGGTGConstruct #13 CTGAAATGTAAAGGCGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACTCCTCCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGATCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAGAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTCTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGAACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGCCCTACAGCTGGTTCTGTTGGAGGCTCTGGACTGGGCACACTGGCCCTGGCTATTGAGGGCAGAATGCCCAAGTCCTGCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTGCTCGGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCTCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCTTGCCACCTTCTCGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCCTGGCAAATGA SEQ ID NO: 43Nucleotide sequence of GCTCAGAATATCACCGCCAGAATCGGCGAGCCCCTGGTGConstruct #16 CTGAAATGTAAAGGCGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGATCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAGAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTCTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGAACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGAGGCTGAGGACTCTCCTCAGCACGCTGTTGAGTGCCCTCCATGTGCTCCTCCAGTTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCACCCGCGAGCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTG GGCAAATGA SEQ ID NO: 44Nucleotide sequence of GCTCAGGAAATCACCGCCAGAATCGGCGAGCCCCTGGTGConstruct #17 CTGAAATGTAAAGGCGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGATCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAGAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTCTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGAACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGAGGCTGAGGACTCTCCTCAGCACGCTGTTGAGTGCCCTCCATGTGCTCCTCCAGTTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGCACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTG GGCAAATGA SEQ ID NO: 45Nucleotide sequence of GCTCAGCAGATCACCGCCAGAATCGGCGAGCCCCTGGTGConstruct #18 CTGAAATGTAAAGGCGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGATCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAGAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTCTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGAACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGAGGCTGAGGACTCTCCTCAGCACGCTGTTGAGTGCCCTCCATGTGCTCCTCCAGTTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGCACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTG GGCAAATGA SEQ ID NO: 46Nucleotide sequence of GCTCAGAACATCACCGCCAGAATCGGCGAGCCCCTGGTGConstruct #19 CTGAAATGTAAAGGCGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACTCTTCCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGATCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAGAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTCTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGAACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGAGGCTGAGGACTCTCCTCAGCACGCTGTTGAGTGCCCTCCATGTGCTCCTCCAGTTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGCACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGCCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTG GGCAAATGA SEQ ID NO: 47Nucleotide sequence of GCTCAGGAAATCACCGCCAGAATCGGCGAGCCCCTGGTGConstruct #20 CTGAAATGTAAAGGCGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACTCTTCCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGATCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAGAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTCTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGAACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGAGGCTGAGGACTCTCCTCAGCACGCTGTTGAGTGCCCTCCATGTGCTCCTCCAGTTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGCACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTG GGCAAATGA SEQ ID NO: 48Nucleotide sequence of GCTCAGCAGATCACCGCCAGAATCGGCGAGCCCCTGGTGConstruct #21 CTGAAATGTAAAGGCGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACTCTTCCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGATCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAGAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTCTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGAACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGAGGCTGAGGACTCTCCTCAGCACGCTGTTGAGTGCCCTCCATGTGCTCCTCCAGTTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGCACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTG GGCAAATGA SEQ ID NO: 49Nucleotide sequence of GCTCAGAACATCACCGCCAGAATCGGCGAGCCCCTGGTGConstruct #22 CTGAAATGTAAAGGCGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTGCTGGCTCCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGATCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAGAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTCTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGAACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGAGGCTGAGGACTCTCCTCAGCACGCTGTTGAGTGCCCTCCATGTGCTCCTCCAGTTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGCACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTG GGCAAATGA SEQ ID NO: 50Nucleotide sequence of GCTCAGGAAATCACCGCCAGAATCGGCGAGCCCCTGGTGConstruct #23 CTGAAATGTAAAGGCGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTGCTGGCTCCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGATCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAGAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTCTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGAACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGAGGCTGAGGACTCTCCTCAGCACGCTGTTGAGTGCCCTCCATGTGCTCCTCCAGTTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGCACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATCCCACTCTAATCCCCAGCCTCACAACAACTACAACACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTG GGCAAATGA SEQ ID NO: 51Nucleotide sequence of GCTCAGCAGATCACCGCCAGAATCGGCGAGCCCCTGGTGConstruct #24 CTGAAATGTAAAGGCGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTGCTGGCTCCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGATCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAGAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTCTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGAACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGAGGCTGAGGACTCTCCTCAGCACGCTGTTGAGTGCCCTCCATGTGCTCCTCCAGTTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGCACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTG GGCAAATGA SEQ ID NO: 5216 AA C-terminal sequence EGFDKVREAEDSPQHM unique to esRAGESEQ ID NO: 53 Construct #9 AQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWRAGE V-C1-C2-shortened KVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEGIFRCQstem-VH8aa-IgG4- AMNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNhinge(S/P-AA)-(IgG4CH2- KVGTCVSEGSYPAGTLSWHLDGKPLVPNEKGVSVKEQTRCH3) (mature protein; RHPETGLFTLQSELMVTPARGGDPRPTFSCSFSPGLPRHlacking the natural leaders RALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGTVTsequence) LTCEVPAQPSPQIHWMKDGVPLPLPPSPVLILPEIGPQDQGTYSCVATHSSHGPQESRAVSISIIEPGEEGPTAGGTLVTVSSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 54 Construct #16ΔKAQNITARIGE PLVLKCKGAP KKPPQRLE RAGEV-C1-C2-V1 stemWKLNTGRTEA WKVLSPQGGG PWDSVARVLP (M/A)-IgG2 lower hinge-NGSLFLPAVG IQDEGIFRCQ AMNRNGKETK (IgG4CH2-CH3)-YTE-AKSNYRVRVYQI PGKPEIVDSA SELTAGVPNK (mature protein; lacking theVGTCVSEGSY PAGTLSWHLD GKPLVPNEKG natural leaders sequence)VSVKEQTRRH PETGLFTLQS ELMVTPARGG DPRPTFSCSF SPGLPRHRAL RTAPIQPRVWEPVPLEEVQL VVEPEGGAVA PGGTVTLTCE VPAQPSPQIH WMKDGVPLPL PPSPVLILPEIGPQDQGTYS CVATHSSHGP QESRAVSISI IEPGEEGPTA GEGFDKVREA EDSPQHAVECPPCAPPVAGG PSVFLFPPKP KDTLYITREP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNAKTKPREEQFN STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQVYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLYSRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLG* SEQ ID NO: 55Construct #12ΔK AQNITARIGE PLVLKCKGAP KKPPQRLE RAGE V-C1-C2-V1 stemWKLNTGRTEA WKVLSPQGGG PWDSVARVLP (M/A)-IgG2 lower hinge-NGSLFLPAVG IQDEGIFRCQ AMNRNGKETK (IgG4CH2-CH3)-ΔKSNYRVRVYQI PGKPEIVDSA SELTAGVPNK (mature protein; lacking theVGTCVSEGSY PAGTLSWHLD GKPLVPNEKG natural leaders sequence)VSVKEQTRRH PETGLFTLQS ELMVTPARGG DPRPTFSCSF SPGLPRHRAL RTAPIQPRVWEPVPLEEVQL VVEPEGGAVA PGGTVTLTCE VPAQPSPQIH WMKDGVPLPL PPSPVLILPEIGPQDQGTYS CVATHSSHGP QESRAVSISI IEPGEEGPTA GEGFDKVREA EDSPQHAVECPPCAPPVAGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNAKTKPREEQFN STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQVYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLYSRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLG* SEQ ID NO: 56Nucleotide sequence of GCTCAGAATATCACCGCCAGAATCGGCGAGCCCCTGGTGConstruct #9 CTGAAATGTAAAGGCGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGATCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAGAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTCTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGAACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGGAACACTGGTCACCGTGTCCTCCGAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTGGGCAAA TGA SEQ ID NO: 57Nucleotide sequence of GCTCAGGAGATCACCGCCAGAATCGGCGAGCCCCTGGTGConstruct #25 CTGAAATGTAAAGGGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGTCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGGGCTGAGGACTCTCCTCAGCACGCTGTTGAGTGCCCTCCATGTGCTCCTCCAGTTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCACCCGCGAGCCTGAATGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCAGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTGGGCAAATGA SEQ ID NO: 58 Nucleotide sequence ofGCTCAGCAGATCACCGCCAGAATCGGCGAGCCCCTGGTG Construct #26CTGAAATGTAAAGGGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGTCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGGGCTGAGGACTCTCCTCAGCACGCTGTTGAGTGCCCTCCATGTGCTCCTCCAGTTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCACCCGCGAGCCTGAATGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCAGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTGGGCAAATGA SEQ ID NO: 59 Nucleotide sequence ofGCTCAGAATATCACCGCCAGAATCGGCGAGCCCCTGGTG Construct #27CTGAAATGTAAAGGGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACTCTTCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGTCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGGGCTGAGGACTCTCCTCAGCACGCTGTTGAGTGCCCTCCATGTGCTCCTCCAGTTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCACCCGCGAGCCTGAATGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCAGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTGGGCAAATGA SEQ ID NO: 60 Nucleotide sequence ofGCTCAGGAGATCACCGCCAGAATCGGCGAGCCCCTGGTG Construct #28CTGAAATGTAAAGGGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACTCCTCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGTCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGGGCTGAGGACTCTCCTCAGCACGCTGTTGAGTGCCCTCCATGTGCTCCTCCAGTTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCACCCGCGAGCCTGAATGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCAGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTGGGCAAATGA SEQ ID NO: 61 Nucleotide sequence ofGCTCAGCAGATCACCGCCAGAATCGGCGAGCCCCTGGTG Construct #29CTGAAATGTAAAGGGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACTCTTCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGTCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGGGCTGAGGACTCTCCTCAGCACGCTGTTGAGTGCCCTCCATGTGCTCCTCCAGTTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCACCCGCGAGCCTGAATGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCAGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTGGGCAAATGA SEQ ID NO: 62 Nucleotide sequence ofGCTCAGAATATCACCGCCAGAATCGGCGAGCCCCTGGTG Construct #30CTGAAATGTAAAGGGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGTCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGGGCTGAGGACTCTCCTCAGCACGCTGAGAGAAAGTGCTGCGTTGAGTGCCCTCCATGTGCTCCTCCAGTTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCCCCGCGAGCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCC TGGGCAAATGA SEQ ID NO: 63Nucleotide sequence of GCTCAGAATATCACCGCCAGAATCGGCGAGCCCCTGGTGConstruct #31 CTGAAATGTAAAGGGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGTCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGGGCTGAGGACTCTCCTCAGCACGCTGTTGAGTGCCCTCCATGTGCTCCTCCAGTGGCTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCACCCGCGAGCCTGAAGTGCCTGCGTGGTGGTGGATGTGTCTCACGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTTCAGAGTGTGTCCGTGCTGACCGTGGTGCATCAGGATTGGCTGAATGGGAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTGCTCCTATCGAAAAGACCATCTCTAAGACCAAGGGACAGCCCCGGGACCTCAGGTGTACACACTGCCACCTAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTAGAACAACTACAAGACCACACCTCCTATGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGCCCTGCACAATCACTACACCC AGAAGTCCCTGTCTCTGTCCCCTGGCAAATGASEQ ID NO: 64 Nucleotide sequence ofGCTCAGAATATCACCGCCAGAATCGGCGAGCCCCTGGTG Construct #32CTGAAATGTAAAGGGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGTCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGGGCTGAGGACTCTCCTCAGCACGCTGTTGAGTGCCCTCCATGTGCTCCTCCAGTTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCACCCGCGAGCCTGAATGACCTGCGTGGTGGTGGATGTGTCTCACGAGGACCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGAACCCCAGGTTTACACCTTGCCACCTTCTCGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCAGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCCTGGCAAATGA SEQ ID NO: 65 Nucleotide sequence ofGCTCAGAATATCACCGCCAGAATCGGCGAGCCCCTGGTG Construct #33CTGAAATGTAAAGGGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGTCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGGGCTGAGGACTCTCCTCAGCACGCTGAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACTCACCCGCGAGCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCTCACGAGGACCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACATACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCTTGCCACCTTCTCGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAAGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGT CCCCTGGCAAATGA SEQ ID NO: 66Nucleotide sequence of GCTCAGAATATCACCGCCAGAATCGGCGAGCCCCTGGTGConstruct #34 CTGAAATGTAAAGGGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGTCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGGGCTGAGGACTCTCCTCAGCACGCTGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACCCCTGTACATCACCCGCGAGCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCTCACGAGGACCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCTTGCCACCTTCTCGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCC TGTCTCTGTCCCCTGGCAAATGASEQ ID NO: 67 Nucleotide sequence ofGCTCAGAATATCACCGCCAGAATCGGCGAGCCCCTGGTG Construct #35CTGAAATGTAAAGGCGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGATCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAGAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTCTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGAACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGAGGCCGAGGATTCTCCTCAGCATGCTGAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGTACATCACCCGGGAGCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAG TCCCTGTCTCTGTCCCTGGGCAAATGASEQ ID NO: 68 Nucleotide sequence ofGCTCAGAATATCACCGCCAGAATCGGCGAGCCCCTGGTG Construct #36CTGAAATGTAAAGGCGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGATCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAGAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTCTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGAACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGAGGCTGAGGACTCTCCTCAGCATGCCGGAACACTGGTCACCGTGTCCTCCGAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGTACATCACCCGGGAGCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCC CTGGGCAAATGA SEQ ID NO: 69Nucleotide sequence of GCTCAGAATATCACCGCCAGAATCGGCGAGCCCCTGGTGConstruct #16ΔK CTGAAATGTAAAGGGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGTCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGGGCTGAGGACTCTCCTCAGCACGCTGTTGAGTGCCCTCCATGTGCTCCTCCAGTTGCTGGTGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCACCCGCGAGCCTGAATGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCAGAGGCCCTGCACAATCACTACA CCCAGAAGTCCCTGTCTCTGTCCCTGGGCTGASEQ ID NO: 70 Nucleotide sequence ofGCTCAGAATATCACCGCCAGAATCGGCGAGCCCCTGGTG Construct #12ΔKCTGAAATGTAAAGGCGCCCCTAAGAAGCCTCCTCAGCGGCTGGAATGGAAGCTGAACACCGGCAGAACCGAGGCCTGGAAAGTGCTGTCTCCTCAAGGCGGAGGCCCTTGGGATTCTGTGGCTAGAGTGCTGCCTAACGGCTCCCTGTTTCTGCCTGCTGTGGGCATCCAGGACGAGGGCATCTTCAGGTGTCAGGCCATGAACCGGAACGGCAAAGAGACAAAGTCCAACTACCGCGTCAGAGTGTATCAGATCCCCGGCAAGCCTGAGATCGTGGACTCTGCCTCTGAACTGACAGCCGGCGTGCCCAACAAAGTGGGCACTTGTGTGTCCGAGGGCAGCTATCCTGCTGGCACCCTGTCTTGGCATCTGGATGGAAAGCCTCTGGTGCCCAACGAGAAAGGCGTGTCCGTGAAAGAGCAGACCAGACGGCATCCTGAGACTGGCCTGTTCACCCTGCAGTCCGAGCTGATGGTTACCCCTGCTAGAGGCGGCGATCCCAGACCTACCTTCAGCTGCTCCTTCTCTCCTGGCCTGCCTCGACATAGAGCCCTGAGAACCGCTCCTATCCAGCCTAGAGTGTGGGAGCCTGTGCCTCTGGAAGAGGTGCAGCTGGTGGTTGAACCTGAAGGCGGAGCTGTTGCTCCTGGCGGAACAGTGACCCTGACCTGTGAAGTTCCCGCTCAGCCCTCTCCACAGATCCACTGGATGAAGGATGGCGTGCCACTGCCTCTGCCTCCATCTCCTGTTCTGATCCTGCCAGAGATCGGCCCTCAGGACCAGGGCACCTATTCTTGTGTGGCTACCCACTCCTCTCACGGCCCTCAAGAGTCTAGAGCCGTGTCCATCTCCATCATCGAGCCTGGCGAGGAAGGACCTACAGCTGGCGAGGGCTTTGACAAAGTGCGCGAGGCTGAGGACTCTCCTCAGCATGCCGTGGAATGCCCTCCTTGTGCTCCTCCTGTGGCTGGCGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAATGGCAAAGAGTATAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGACGGCAGCTTCTTTCTGTACTCCCGCCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCCCTG GGCTGA SEQ ID NO: 71Short Stem sequence (#9) HSSHGPQESRAVSISIIEPGEEGPTAG SEQ ID NO: 72V1 stem sequence HSSHGPQESRAVSISIIEPGEEGPTAGEGFDKVREAEDS PQHASEQ ID NO: 73 C-terminal 13 amino acids of SVGGSGLGTLALA RAGE stemSEQ ID NO: 74 esRAGE (sequence of the AQNITARI GEPLVLKCKG APKKPPQRLEmature protein; lacking the WKLNTGRTEA WKVLSPQGGG PWDSVARVLPnatural leader sequence) NGSLFLPAVG IQDEGIFRCQ AMNRNGKETKSNYRVRVYQI PGKPEIVDSA SELTAGVPNK VGTCVSEGSY PAGTLSWHLD GKPLVPNEKGVSVKEQTRRH PETGLFTLQS ELMVTPARGG DPRPTFSCSF SPGLPRHRAL RTAPIQPRVWEPVPLEEVQL VVEPEGGAVA PGGTVTLTCE VPAQPSPQIH WMKDGVPLPL PPSPVLILPEIGPQDQGTYS CVATHSSHGP QESRAVSISI IEPGEEGPTA GEGFDKVREA EDSPQHM

1. An isolated polypeptide comprising: (a) a first domain wherein saidfirst domain has an amino acid sequence at least 97% identical to thesequence of SEQ ID NO:74; and (b) a second domain comprising a fragmentof a Fc region of an immunoglobulin, wherein the carboxy terminus ofsaid first domain is coupled to the amino terminus of said second domainby a peptide linkage.
 2. The isolated polypeptide of claim 1, whereinsaid polypeptide is resistant to cleavage by a disintegrin andmetalloproteinase 10 (ADAM10).
 3. The isolated polypeptide of claim 1,wherein said polypeptide is at least 15% more resistant to cleavage byat least one of ADAM10, matrix metalloproteinase 9 (MMP9), and trypsinas compared to a polypeptide having the sequence of SEQ ID NO:
 5. 4. Theisolated polypeptide of claim 1, wherein said polypeptide is at least15% more resistant to degradation in human serum as compared to apolypeptide comprising the sequence of SEQ ID NO: 5, wherein the percentresistance equals the difference between the fraction of peptide thatremains full length following incubation in human serum for a definedtime period compared to a control peptide treated for the same time andunder the same conditions.
 5. The isolated polypeptide of claim 1,wherein said polypeptide has increased thermal stability of at least 5°C. as compared to a polypeptide having the sequence of SEQ ID NO:
 5. 6.The isolated polypeptide of claim 1, wherein said polypeptidespecifically binds an advanced glycation endproduct (AGE).
 7. Theisolated polypeptide of claim 1, wherein said polypeptide specificallybinds HMGB1 (Amphoterin).
 8. The isolated polypeptide of claim 1,wherein said polypeptide specifically binds at least one of the groupconsisting of: S100A1, S100A2, S100A4 (metastasin), S100A5, S100A6,S100A7 (psoriasin), S100A8/9, S100A11, S100A12, S100B, S100P,lipopolysaccharide (LPS), oxidized low-density lipoprotein (oxLDL),CD11b (MAC1), phosphatidyl serine, C3 a, S100P, S100G, S100Z,carbonylated proteins, malondialdehyde (MDA), laminin, type I Collagen,type IV Collagen, CAPZA1, CAPZA2, DDOST, LGALS3, MAPK1, MAPK3, PRKCSH,S100A4, S100A5, S100A6, S100A8, S100A9, S100P, and SAA1.
 9. The isolatedpolypeptide of claim 1, wherein said polypeptide specifically bindsamyloidbeta.
 10. The isolated polypeptide of claim 1, wherein saidpolypeptide is a dimer of a polypeptide having the sequence of SEQ IDNO:
 74. 11. The isolated polypeptide of claim 1, wherein said firstdomain comprises at least one asparagine residue linked to a glycan. 12.The isolated polypeptide of claim 1, wherein said first domain comprisesan amino acid substitution at one or more of amino acid residues 3 or 59of SEQ ID NO:74.
 13. The isolated polypeptide of claim 12, wherein saidamino acid substitution is a substitution with glutamic acid orglutamine at amino acid residue
 3. 14. The isolated polypeptide of claim12, wherein said amino acid substitution is a substitution with alanine,glutamic acid, or glutamine at amino acid residue
 59. 15. The isolatedpolypeptide of claim 1, wherein said first domain comprises an aminoacid substitution at amino acid residue 60 of SEQ ID NO:74.
 16. Theisolated polypeptide of claim 1, wherein said first domain has thesequence set forth in SEQ ID NO:
 74. 17. The isolated polypeptide ofclaim 15, wherein said amino acid substitution is a substitution withserine.
 18. The isolated polypeptide of claim 1, wherein said Fcfragment comprises CH2 and CH3 domains of a human IgG.
 19. An isolatedpolypeptide comprising a RAGE polypeptide coupled to a Fc region of animmunoglobulin, wherein the carboxy terminus of said RAGE polypeptide iscoupled to the amino terminus of said immunoglobulin Fc region by apeptide linkage and wherein said RAGE polypeptide has the sequence ofSEQ ID NO:
 2. 20. A pharmaceutical composition for treating aRAGE-mediated disorder comprising the isolated polypeptide of claim 1.